Treatment of fibrosis with interleukin-11 receptor alpha antibody

ABSTRACT

Aspects of the disclosure relate to the treatment, prevention or alleviation of conditions such as fibrosis in a subject. In some embodiments, the treatment, prevention or alleviation of fibrosis in a subject through the administration of an agent capable of inhibiting the action of Interleukin 11 (IL-11) is disclosed.

RELATED APPLICATIONS

The present application is a continuation application of U.S.application Ser. No. 15/988,463, filed May 24, 2018, which is adivisional application of U.S. application Ser. No. 15/381,622, filedDec. 16, 2016, now U.S. Pat. No. 10,035,852, which claims priority under35 USC § 119(a)-(d) to United Kingdom Application No. 1522186.4, filedDec. 16, 2015. The entire contents of the aforementioned applicationsare hereby incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to the diagnosis and treatment ofconditions such as fibrosis.

BACKGROUND TO THE INVENTION

Fibrosis is an essential process that is a critical part of woundhealing. Excessive fibrosis is common in many rare and common diseaseconditions and is important in disease pathogenesis. Diseasescharacterized by excessive fibrosis include but are not restricted to:systemic sclerosis, scleroderma, hypertrophic cardiomyopathy, dilatedcardiomyopathy (DCM), atrial fibrillation, ventricular fibrillation,myocarditis, liver cirrhosis, kidney diseases, diseases of the eye,asthma, cystic fibrosis, arthritis and idiopathic pulmonary fibrosis.Despite the large impact on human health, therapeutic and diagnosticapproaches to fibrosis are still an unmet medical need.

The real physiological role of Interleukin 11 (IL-11) remains unclear.IL-11 has been most strongly linked with activation of haematopoeticcells and with platelet production but also found to be pro- as well asanti-inflammatory, pro-angiogenic and important for neoplasia. It isknown that TGFβ1 or tissue injury can induce IL-11 expression (Zhu, M.et al. IL-11 Attenuates Liver Ischemia/Reperfusion Injury (IRI) throughSTAT3 Signaling Pathway in Mice. PLOS ONE 10, (2015); Yashiro, R. et al.Transforming growth factor-beta stimulates interleukin-11 production byhuman periodontal ligament and gingival fibroblasts. J. Clin.Periodontol. 33, 165-71 (2006); Obana, M. et al. Therapeutic activationof signal transducer and activator of transcription 3 by interleukin-11ameliorates cardiac fibrosis after myocardial infarction. Circulation121, 684-91 (2010); Tang, W., Yang, L., Yang, Y. C., Leng, S. X. &Elias, J. A. Transforming growth factor-beta stimulates interleukin-11transcription via complex activating protein-1-dependent pathways. J.Biol. Chem. 273, 5506-13 (1998)).

The role for IL-11 in fibrosis is not clear from the publishedliterature. IL-11 is thought to be important for fibrosis andinflammation in the lung (Tang, W. et al. Targeted expression of IL-11in the murine airway causes lymphocytic inflammation, bronchialremodeling, and airways obstruction. J. Clin. Invest. 98, 2845-53(1996)) and its expression level is correlated with collagen levels inthe skin (Toda, M. et al. Polarized in vivo expression of IL-11 andIL-17 between acute and chronic skin lesions. Journal of Allergy andClinical Immunology 111, 875-881 (2003)) and the respiratory system(Molet, S., Hamid, Q. & Hamilos, D. IL-11 and IL-17 expression in nasalpolyps: Relationship to collagen deposition and suppression byintranasal fluticasone propionate. The Laryngoscope 113, (2003);Minshall et al. IL-11 expression is increased in severe asthma:association with epithelial cells and eosinophils. The Journal ofallergy and clinical immunology 105, (2000)).

However, the majority of studies suggest that IL-11 is anti-fibrotic: inthe heart (Obana, M. et al. Therapeutic activation of signal transducerand activator of transcription 3 by interleukin-11 ameliorates cardiacfibrosis after myocardial infarction. Circulation 121, 684-91 (2010);Obana, M. et al. Therapeutic administration of IL-11 exhibits thepostconditioning effects against ischemia-reperfusion injury via STAT3in the heart. American Journal of Physiology. Heart and circulatoryphysiology 303, H569-77 (2012)) and kidney (Stangou, M. et al. Effect ofIL-11 on glomerular expression of TGF-beta and extracellular matrix innephrotoxic nephritis in Wistar Kyoto rats. Journal of nephrology 24,106-11 (2011); Ham, A. et al. Critical role of interleukin-11 inisoflurane-mediated protection against ischemic acute kidney injury inmice. Anesthesiology 119, 1389-401 (2013)) and anti-inflammatory inseveral tissues and chronic inflammatory diseases (Trepicchio & Dorner.The therapeutic utility of Interleukin-11 in the treatment ofinflammatory disease. (1998). doi:10.1517/13543784.7.9.1501). Themolecular mode of action of IL-11 in general, is thought to beregulation of RNA expression of mRNA levels via STAT3-mediatedtranscription (Zhu, M. et al. IL-11 Attenuates LiverIschemia/Reperfusion Injury (IRI) through STAT3 Signaling Pathway inMice. PLOS ONE 10, (2015)).

SUMMARY OF THE INVENTION

One aspect of the present invention concerns the treatment, preventionor alleviation of fibrosis in a subject in need of treatment through theadministration of an agent capable of inhibiting the action ofInterleukin 11 (IL-11). The inventors have identified IL-11 to have apro-fibrotic action. The present invention is particularly concernedwith inhibiting the pro-fibrotic action of IL-11. Embodiments of theinvention concern inhibition or prevention of the IL-11 mediatedpro-fibrotic signal, e.g. as mediated by binding of IL-11 to an IL-11receptor.

In some embodiments an agent capable of inhibiting the action of IL-11may prevent or reduce the binding of IL-11 to an IL-11 receptor.

In some embodiments an agent capable of inhibiting the action of IL-11may bind IL-11 to form a complex comprising the agent and IL-11. Thecomplex may be a non-covalent or covalent complex. In some embodiments,the formation of the agent:IL-11 complex may prevent or reduce theability of IL-11 to bind to an IL-11 receptor. In some embodiments suchprevention or reduction may be the result of a reduction of theproductive binding of IL-11 to an IL-11 receptor, i.e. reduction in theability of IL-11 to initiate IL-11 receptor mediated signaling. In someembodiments formation of the agent:IL-11 complex may sequester IL-11away from the IL-11 receptor, thereby preventing or reducing the contactof IL-11 with an IL-11 receptor and/or preventing or reducing the amountof IL-11 available for binding to an IL-11 receptor. In some embodimentsthe agent may be a decoy receptor.

In some embodiments an agent capable of inhibiting the action of IL-11may bind to an IL-11 receptor. An agent that binds an IL-11 receptor mayprevent or reduce the ability of IL-11 to bind to an IL-11 receptor(IL-11R).

Another aspect of the present invention concerns the treatment,prevention or alleviation of fibrosis in a subject in need of treatmentthrough the administration of an agent capable of preventing or reducingthe expression of IL-11 or an IL-11 receptor (IL-11R).

In one aspect of the present invention an agent capable of inhibitingthe action of Interleukin 11 (IL-11) for use in a method of treating orpreventing fibrosis is provided.

In another aspect of the present invention the use of an agent capableof inhibiting the action of IL-11 in the manufacture of a medicament foruse in a method of treating or preventing fibrosis is provided.

In another aspect of the present invention a method of treating orpreventing fibrosis is provided, the method comprising administering toa subject in need of treatment a therapeutically effective amount of anagent capable of inhibiting the action of IL-11.

In some embodiments the agent capable of inhibiting the action of IL-11is an agent capable of preventing or reducing the binding of IL-11 to anIL-11 receptor.

In some embodiments the agent capable of inhibiting the action of IL-11is an IL-11 binding agent. IL-11 binding agents may be selected from thegroup consisting of: an antibody, polypeptide, peptide, oligonucleotide,aptamer or small molecule. In some embodiments the IL-11 binding agentis an antibody. In some embodiments the IL-11 binding agent is a decoyreceptor.

In some embodiments the agent capable of inhibiting the action of IL-11is an IL-11 receptor (IL-11R) binding agent. IL-11R binding agents maybe selected from the group consisting of: an antibody, polypeptide,peptide, oligonucleotide, aptamer or small molecule. In some embodimentsthe IL-11R binding agent is an antibody.

In another aspect of the present invention an agent capable ofpreventing or reducing the expression of IL-11 or IL-11R for use in amethod of treating or preventing fibrosis is provided.

In another aspect of the present invention the use of an agent capableof preventing or reducing the expression of IL-11 or IL-11R in themanufacture of a medicament for use in a method of treating orpreventing fibrosis is provided.

In another aspect of the present invention a method of treating orpreventing fibrosis is provided, the method comprising administering toa subject in need of treatment a therapeutically effective amount of anagent capable of preventing or reducing the expression of IL-11 orIL-11R.

In some embodiments the agent capable of preventing or reducing theexpression of IL-11 or IL-11R is a small molecule or oligonucleotide.

In some embodiments the fibrosis to be treated or prevented is fibrosisof the heart, liver or kidney. In some embodiments the fibrosis to betreated or prevented is fibrosis of the eye. In some embodiments thefibrosis is in the heart and is associated with dysfunction of themusculature or electrical properties of the heart, or thickening of thewalls or valves of the heart. In some embodiments the fibrosis is in theliver and is associated with chronic liver disease or liver cirrhosis.In some embodiments the fibrosis is in the kidney and is associated withchronic kidney disease.

In some embodiments the method of treating or preventing comprisesadministering a said agent to a subject in which IL-11 or IL-11Rexpression is upregulated. In some embodiments the method of treating orpreventing comprises administering a said agent to a subject in whichIL-11 or IL-11R expression has been determined to be upregulated. Insome embodiments the method of treating or preventing comprisesdetermining whether IL-11 or IL-11R expression is upregulated in thesubject and administering a said agent to a subject in which IL-11 orIL-11R expression is upregulated.

In another aspect of the present invention a method of determining thesuitability of a subject for the treatment or prevention of fibrosiswith an agent capable of inhibiting the action of IL-11 is provided, themethod comprising determining, optionally in vitro, whether IL-11 orIL-11R expression is upregulated in the subject.

In another aspect of the present invention a method of selecting asubject for the treatment or prevention of fibrosis with an agentcapable of inhibiting the action of IL-11 is provided, the methodcomprising determining, optionally in vitro, whether IL-11 or IL-11Rexpression is upregulated in the subject.

In another aspect of the present invention a method of diagnosingfibrosis or a risk of developing fibrosis in a subject is provided, themethod comprising determining, optionally in vitro, the upregulation ofIL-11 or IL-11R in a sample obtained from the subject.

In some embodiments the method is a method of confirming a diagnosis offibrosis in a subject suspected of having fibrosis.

In some embodiments the method further comprises selecting the subjectfor treatment with an agent capable of inhibiting the action of IL-11 orwith an agent capable of preventing or reducing the expression of IL-11or IL-11R.

In another aspect of the present invention a method of providing aprognosis for a subject having, or suspected of having fibrosis, isprovided, the method comprising determining, optionally in vitro,whether IL-11 or IL-11R is upregulated in a sample obtained from thesubject and, based on the determination, providing a prognosis fortreatment of the subject with an agent capable of inhibiting the actionof IL-11 or with an agent capable of preventing or reducing theexpression of IL-11 or IL-11R.

The method may further comprise selecting a subject determined to haveupregulated IL-11 or IL-11R for treatment with an agent capable ofinhibiting the action of IL-11 or with an agent capable of preventing orreducing the expression of IL-11 or IL-11R.

In another aspect of the present invention a method of diagnosingfibrosis or a risk of developing fibrosis in a subject is provided, themethod comprising determining, optionally in vitro, one or more geneticfactors in the subject that are predictive of upregulation of IL-11 orIL-11R expression, or of upregulation of IL-11 or IL-11R activity.

In some embodiments the method is a method of confirming a diagnosis offibrosis in a subject suspected of having fibrosis.

In some embodiments the method further comprises selecting the subjectfor treatment with an agent capable of inhibiting the action of IL-11 orwith an agent capable of preventing or reducing the expression of IL-11or IL-11R.

In another aspect of the present invention a method of providing aprognosis for a subject having, or suspected of having, fibrosis, isprovided, the method comprising determining, optionally in vitro, one ormore genetic factors in the subject that are predictive of upregulationof IL-11 or IL-11R expression, or of upregulation of IL-11 or IL-11Ractivity.

The following numbered paragraphs (paras) describe further aspects andembodiments of the present invention:

1. An agent capable of inhibiting the action of Interleukin 11 (IL-11)for use in a method of treating or preventing fibrosis.

2. Use of an agent capable of inhibiting the action of Interleukin 11(IL-11) in the manufacture of a medicament for use in a method oftreating or preventing fibrosis.

3. A method of treating or preventing fibrosis, the method comprisingadministering to a subject in need of treatment a therapeuticallyeffective amount of an agent capable of inhibiting the action ofInterleukin 11 (IL-11).

4. The agent for use in a method of treating or preventing fibrosisaccording to para 1, use according to para 2 or method according to para3, wherein the agent is an agent capable of preventing or reducing thebinding of IL-11 to an IL-11 receptor.

5. The agent for use in a method of treating or preventing fibrosisaccording to para 1 or 4, use according to para 2 or 4, or methodaccording to para 3 or 4, wherein the agent is an IL-11 binding agent.

6. The agent for use in a method of treating or preventing fibrosis, useor method according to para 5, wherein the IL-11 binding agent isselected from the group consisting of: an antibody, polypeptide,peptide, oligonucleotide, aptamer or small molecule.

7. The agent for use in a method of treating or preventing fibrosis, useor method according to para 5, wherein the IL-11 binding agent is anantibody.

8. The agent for use in a method of treating or preventing fibrosis, useor method according to para 5, wherein the IL-11 binding agent is adecoy receptor.

9. The agent for use in a method of treating or preventing fibrosisaccording to para 1 or 4, use according to para 2 or 4, or methodaccording to para 3 or 4, wherein the agent is an IL-11 receptor(IL-11R) binding agent.

10. The agent for use in a method of treating or preventing fibrosis,use or method according to para 9, wherein the IL-11R binding agent isselected from the group consisting of: an antibody, polypeptide,peptide, oligonucleotide, aptamer or small molecule.

11. The agent for use in a method of treating or preventing fibrosis,use or method according to para 9, wherein the IL-11R binding agent isan antibody.

12. An agent capable of preventing or reducing the expression ofInterleukin 11 (IL-11) or an Interleukin 11 receptor (IL-11R) for use ina method of treating or preventing fibrosis.

13. Use of an agent capable of preventing or reducing the expression ofInterleukin 11 (IL-11) or an Interleukin 11 receptor (IL-11R) in themanufacture of a medicament for use in a method of treating orpreventing fibrosis.

14. A method of treating or preventing fibrosis, the method comprisingadministering to a subject in need of treatment a therapeuticallyeffective amount of an agent capable of preventing or reducing theexpression of Interleukin 11 (IL-11) or an Interleukin 11 receptor(IL-11R).

15. The agent for use in a method of treating or preventing fibrosisaccording to para 12, use according to para 13 or method according topara 14, wherein the agent is a small molecule or oligonucleotide.

16. The agent for use in a method of treating or preventing fibrosis,use or method according to any one of the preceding paras, wherein thefibrosis is fibrosis of the heart, liver, kidney or eye.

17. The agent for use in a method of treating or preventing fibrosis,use or method according to any one of the preceding paras, wherein thefibrosis is in the heart and is associated with dysfunction of themusculature or electrical properties of the heart, or thickening of thewalls or valves of the heart.

18. The agent for use in a method of treating or preventing fibrosis,use or method according to any one of the preceding paras, wherein thefibrosis is in the liver and is associated with chronic liver disease orliver cirrhosis.

19. The agent for use in a method of treating or preventing fibrosis,use or method according to any one of the preceding paras, wherein thefibrosis is in the kidney and is associated with chronic kidney disease.

20. The agent for use in a method of treating or preventing fibrosis,use or method according to any one of the preceding paras, wherein thefibrosis is in the eye and is retinal fibrosis, epiretinal fibrosis, orsubretinal fibrosis.

21. The agent for use in a method of treating or preventing fibrosis,use or method according to any one of the preceding paras, wherein themethod of treating or preventing comprises administering said agent to asubject in which IL-11 or IL-11R expression is upregulated.

22. The agent for use in a method of treating or preventing fibrosis,use or method according to any one of the preceding paras, wherein themethod of treating or preventing comprises administering said agent to asubject in which IL-11 or IL-11R expression has been determined to beupregulated.

23. The agent for use in a method of treating or preventing fibrosis,use or method according to any one of the preceding paras, wherein themethod of treating or preventing comprises determining whether IL-11 orIL-11R expression is upregulated in the subject and administering saidagent to a subject in which IL-11 or IL-11R expression is upregulated.

24. A method of determining the suitability of a subject for thetreatment or prevention of fibrosis with an agent capable of inhibitingthe action of Interleukin 11 (IL-11), the method comprising determining,optionally in vitro, whether IL-11 or an Interleukin 11 receptor(IL-11R) expression is upregulated in the subject.

25. A method of selecting a subject for the treatment or prevention offibrosis with an agent capable of inhibiting the action of Interleukin11 (IL-11), the method comprising determining, optionally in vitro,whether IL-11 or an Interleukin 11 receptor (IL-11R) expression isupregulated in the subject.

26. A method of diagnosing fibrosis or a risk of developing fibrosis ina subject, the method comprising determining, optionally in vitro, theupregulation of Interleukin 11 (IL-11) or an Interleukin 11 receptor(IL-11R) in a sample obtained from the subject.

27. The method of para 26, wherein the method is a method of confirminga diagnosis of fibrosis in a subject suspected of having fibrosis.

28. The method of para 26 or 27, wherein the method further comprisesselecting the subject for treatment with an agent capable of inhibitingthe action of IL-11 or with an agent capable of preventing or reducingthe expression of IL-11 or IL-11R.

29. A method of providing a prognosis for a subject having, or suspectedof having fibrosis, the method comprising determining, optionally invitro, whether Interleukin 11 (IL-11) or an Interleukin 11 receptor(IL-11R) is upregulated in a sample obtained from the subject and, basedon the determination, providing a prognosis for treatment of the subjectwith an agent capable of inhibiting the action of IL-11 or with an agentcapable of preventing or reducing the expression of IL-11 or IL-11R.

30. The method of para 29, wherein the method further comprisesselecting a subject determined to have upregulated IL-11 or IL-11R fortreatment with an agent capable of inhibiting the action of IL-11 orwith an agent capable of preventing or reducing the expression of IL-11or IL-11R.

31. A method of diagnosing fibrosis or a risk of developing fibrosis ina subject, the method comprising determining, optionally in vitro, oneor more genetic factors in the subject that are predictive ofupregulation of Interleukin 11 (IL-11) or an Interleukin 11 receptor(IL-11R) expression, or of upregulation of IL-11 or IL-11R activity.

32. The method of para 31, wherein the method is a method of confirminga diagnosis of fibrosis in a subject suspected of having fibrosis.

33. The method of para 32 or 32, wherein the method further comprisesselecting the subject for treatment with an agent capable of inhibitingthe action of IL-11 or with an agent capable of preventing or reducingthe expression of IL-11 or IL-11R.

34. A method of providing a prognosis for a subject having, or suspectedof having, fibrosis, the method comprising determining, optionally invitro, one or more genetic factors in the subject that are predictive ofupregulation of Interleukin 11 (IL-11) or an Interleukin 11 receptor(IL-11R) expression, or of upregulation of IL-11 or IL-11R activity.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments and experiments illustrating the principles of the inventionwill now be discussed with reference to the accompanying figures inwhich:

FIGS. 1A, 1B, 1C and 1D. TGFβ1 stimulation upregulates IL-11 infibroblasts. Primary fibroblasts were derived from human atrial tissueof 80 individuals and incubated for 24 h with and without TGFβ1 (5ng/ml). (FIG. 1A) Chart showing IL-11 was the most upregulated gene inTGFβ1 stimulated fibroblasts compared to 11,433 expressed genes(FPKM≥0.5). (FIG. 1B) Chart showing IL-11 expression significantlyincreased more than 8-fold on average after fibroblast activation withTGFβ1 (FDR=9.1×10⁻¹²⁵). (FIG. 1C) Chart showing RT-qPCR confirmed IL-11RNA expression-based fold changes (TGFB1+/TGFB1−; R²=0.94) and (FIG. 1D)Chart showing ELISA detected a significant increase in IL-11 proteinsecreted by stimulated fibroblasts.

FIGS. 2A, 2B, 2C and 2D. Human atrial fibroblasts were incubated eitherwith 5 ng/ml TGFβ1 or 5 ng/ml IL-11 for 24 hours. Charts show cellstaining for (FIG. 2A) α-SMA (myofibroblasts), (FIG. 2B) EdU(proliferation), (FIG. 2C) collagen and (FIG. 2D) periostin to identifymyofibroblasts and highly proliferative cells and to quantify theproduction of extracellular matrix proteins. IL-11 was found to increasethe myofibroblast ratio and induce the production of collagen andperiostin at a similar rate as TGFβ1 signaling. This experiment wasrepeated a number of times with similar results.

FIGS. 3A, 3B, 3C and 3D. Inhibition of IL-11 with a neutralizingantibody prevents TGFβ1-induced fibrosis. Human atrial fibroblasts werestimulated with TGFβ1 (5 ng/ml), TGFβ1 and an antibody against IL-11 orTGFβ1 and an isotype control. Charts and photographs show cell stainedafter 24 hours for (FIG. 3A) α-SMA, (FIG. 3B) EdU, (FIG. 3C) collagenand (FIG. 3D) periostin to identify myofibroblasts and highlyproliferative cells and to quantify the production of extracellularmatrix proteins. Fluorescence was quantified on the Operetta platformfor up to 21 fields per condition. This experiment was repeated withfibroblasts derived from different individuals with similar results. Inthe presence of an antibody blocking IL-11, TGFβ1-stimulated fibroblastshave a decreased ratio of myofibroblasts, are less proliferative andexpress less collagen and periostin compared to control cells. Thisshows that IL-11 is an essential component of TGFβ1 signaling pathwayacting in an autocrine and/or paracrine feed forward fashion and itsinhibition reduces the pro-fibrotic effects of this key regulator offibrosis in humans.

FIGS. 4A and 4B. TGFβ1 stimulation upregulates IL-11 in fibroblasts.Primary fibroblasts were derived from human atrial tissue of 80individuals and incubated for 24 h with and without TGFβ1 (5 ng/ml).(FIG. 4A) Chart showing IL-11 was the most upregulated RNA transcript inTGFβ1 stimulated fibroblasts compared to 11,433 expressed genes(FPKM≥0.5) across the genome as assessed by global transcriptomeprofiling. (FIG. 4B) Chart showing IL-11 expression in non-stimulated(TGF-β−) and stimulated (TGF-β+) primary human fibroblasts compared toall human tissues as assessed by the GTEX project (Consortium, Gte.Human genomics. The Genotype-Tissue Expression (GTEx) pilot analysis:multitissue gene regulation in humans. Science (New York, N.Y.) 348,(2015)) reveals high specificity of elevated IL-11 levels to fibroblastsand specifically activated fibroblasts, the signature of which is notappreciated at the level of the whole organ that contains multiple celltypes and few, IL11-expressing, fibroblasts.

FIGS. 5A, 5B, 5C and 5D. IL-11 acts as an autocrine factor onfibroblasts and induces its own expression via translational regulationalone. Primary fibroblasts were stimulated with TGF-β for 24 hours.(FIG. 5A) Chart showing IL-11 RNA expression increased significantly(FDR=9.1×10⁻¹²⁵) more than 8-fold on average across 80 individuals.(FIG. 5B) Chart showing results of an ELISA assay confirming asignificant increase in IL-11 protein secreted by stimulated fibroblasts(t-test). (FIG. 5C) Chart showing incubation of primary fibroblasts withIL-11 does not increase IL-11 RNA levels (RT-qPCR). (FIG. 5D) Chartshowing incubation of primary fibroblasts with IL-11 induces IL-11protein secretion significantly (Dunnett) as detected by ELISA. AdjustedP-values are given as **** P<0.0001.

FIGS. 6A, 6B, 6C, 6D, 6E and 6F. IL-11 drives proliferation andactivation of fibroblasts as well as extracellular matrix production andis required for the TGFβ1-mediated fibrotic response. Cardiacfibroblasts derived from 3 individuals were incubated for 24 h withTGFβ1 (5 ng/ml), IL-11 (5 ng/ml) or TGFβ1 and a neutralizingIL-11/control antibody. Charts and photographs show results of cellstaining following incubation for (FIG. 6A) α-SMA content to estimatethe fraction of myofibroblasts, (FIG. 6B) EdU to track activelyproliferating cells (FIG. 6C) Periostin to estimate ECM production.Fluorescence was measured with the Operetta platform for 14 fieldsacross 2 wells for each patient. Charts also show the secretion offibrosis markers IL-6 (FIG. 6D), TIMP1 (FIG. 6E) and MMP2 (FIG. 6F) asassessed via ELISA. Fluorescence was normalized to the control groupwithout stimulation and the mean with standard deviation is plotted.IL-11 induces a fibrotic response at similar levels as TGFβ1 andinhibition of IL-11 rescues the TGFβ1 phenotype on the protein level.Adjusted P-values are given as * P<0.05, ** P<0.01, *** P<0.001 or ****P<0.0001 of experimental groups compared to unstimulated cells(Dunnett). Outliers were removed (ROUT, Q=2%).

FIGS. 7A, 7B and 7C. IL-11 promotes collagen protein synthesis andstalls the pro-fibrotic effect of TGFβ1 at the RNA level. Cardiacfibroblasts derived from 3 individuals were incubated for 24 h withTGFβ1 (5 ng/ml), IL-11 (5 ng/ml) or TGFβ1 and a neutralizing IL-11antibody. Following incubation (FIG. 7A) Chart showing results followingincubation of cell staining for collagen using the Operetta assay;florescence was quantified as described above for FIG. 6, (FIG. 7B)Chart showing secreted collagen levels assessed with a Sirius Redstaining and (FIG. 7C) Chart showing collagen RNA levels measured byRT-qPCR. IL-11 induces a fibrotic response at similar levels as TGFβ1only at the protein level. Higher expression of Collagen RNA transcriptsby TGFβ1 did not lead to increased protein production if IL-11 wasneutralized with an antibody. Adjusted P-values are given as * P<0.05,*** P<0.001 or **** P<0.0001 of experimental groups compared tounstimulated cell control group (Dunnett).

FIGS. 8A, 8B, 8C, 8D and 8E. IL-11 is a fibrosis marker and activatoracross multiple tissues. Expression of IL-11 can be induced by a diverseset of upstream pro-fibrotic stimulants in addition to TGFβ1. (FIG. 8A)Chart showing effect of TGFβ1 on IL-11 expression. (FIG. 8B) Chartshowing ET-1 (Endothelin) upregulates IL-11 in hepatic and pulmonaryfibroblasts; (FIG. 8C) Chart showing PDGF (platelet derived growthfactor) induces IL-11 expression in renal fibroblasts. IL-11 RNA levelswere measured by RT-qPCR; adjusted P-values are given as * P<0.05, **P<0.01 or **** P<0.0001 (Dunnett). To investigate the systemic effect ofIL-11, saline only (grey) or recombinant IL-11 (black) was injected 6times a week in C57BL/6 mice (200 μg/kg). Collagen content in tissue wasassessed with a hydroxyproline assay (QuickZyme) on the protein leveland the results are shown in chart (FIG. 8D). Tissues of animals treatedwith rIL-11 have higher collagen protein content than controls (ANOVA;p=0.012). (FIG. 8E) Photographs of western blot showing αSMA levels areincreased in the kidney and heart of IL-11 treated mice, indicating thepresence of myofibroblasts.

FIG. 9. Diagram illustrating role of IL-11 as an essential regulator ofthe fibrotic response. IL-11 is an essential regulator required for thefibrotic response. In response to tissue damage or chronic inflammation,cytokines such as TGFβ1, ET-1 or PDGF are released to upregulate thetranscription of fibrosis marker genes. The autocrine agent IL-11 isthen produced in response to these upstream stimuli to ensure efficienttranslation of upregulated transcripts into functionally relevantproteins in a cell-specific manner. Inhibition of IL-11 blocks thesynthesis of key extracellular matrix and myofibroblast proteins andprevents the pro-fibrotic action of a diverse set of upstream stimuli.

FIG. 10. Inhibition of IL-11 stops collagen protein synthesis inresponse to pro-fibrotic cytokines ANG2 (Angiotensin II), PDGF and ET-1.Cardiac fibroblasts were incubated for 24 h with ANG2, PDGF or ET-1 anda neutralizing IL-11 antibody. Following incubation cells were stainedfor collagen and florescence was quantified. These stimuli induce afibrotic response at similar levels to TGFβ1. However, collagenexpression is not increased if IL-11 is neutralized with an antibody.P-values are given as: **** P<0.0001 (t-test).

FIG. 11. Nucleotide sequence of human IL-11, taken from Genbankaccession number gi|391353405|ref|NM_000641.3 (Homo sapiens interleukin11 (IL11), transcript variant 1, Mrna) [SEQ ID NO:1]. Underlinedsequence encodes IL-11 mRNA. Shaded sequences were used for design ofIL-11 knockdown siRNA and are shown separately as SEQ ID NOs 2 to 5. SEQID NOs 3 and 4 overlap with each other within SEQ ID NO:1.

FIG. 12. Nucleotide sequence of human IL-11Rα, taken from Genbankaccession number gi|975336|gb|U32324.1|HSU32324 (Human interleukin-11receptor alpha chain mRNA, complete cds) [SEQ ID NO:6]. Underlinedsequence encodes IL-11Rα mRNA. Shaded sequences were used for design ofIL-11Rα knockdown siRNA and are shown separately as SEQ ID NOs 7 to 10.

FIG. 13. Table showing siRNA sequences [SEQ ID NOs 11 to 14] forknockdown of IL-11.

FIG. 14. Table showing siRNA sequences [SEQ ID NOs 15 to 18] forknockdown of IL-11Rα.

FIG. 15. Chart showing siRNA knockdown of IL-11Rα in HEK cells.

FIG. 16. Graph showing read depth for whole transcriptome sequencing ofhuman atrial fibroblasts from 160 individuals with and withoutstimulation with TGFβ1.

FIGS. 17A, 17B, 17C, 17D and 17E. Graphs showing expression ofendothelial, cardiomyocyte and fibroblast marker genes as determined byRNA-seq of the tissue of origin (human atrial tissues samples, n=8) andprimary, unstimulated fibroblast cultures. (FIG. 17A) PECAM1, (FIG. 17B)MYH6 (FIG. 17C) TNNT2, (FIG. 17D) COL1A2, and (FIG. 17E) ACTA2.

FIGS. 18A, 18B, 18C, 18D and 18E. Graphs showing upregulation of IL-11expression in fibroblasts in response to stimulation with TGFβ1. (FIG.18A and FIG. 18B) Graphs showing fold change in gene expression infibrosis; IL-11 is the most upregulated gene in response to TGFβ1treatment. (FIG. 18C) IL-11 secretion by fibroblasts in response tostimulation with TGFβ1. (FIG. 18D) Comparison of IL-11 gene expressionin tissues of healthy individuals and in atrial fibroblasts, with orwithout TGFβ1 stimulation. (FIG. 18E) Correspondence of fold change inIL-11 expression as determined by RNA-seq vs. qPCR.

FIGS. 19A, 19B, 19C and 19D. Graphs showing induction of IL-11 secretionin primary fibroblasts by various profibrotic cytokines, as determinedby ELISA. (FIG. 19A) TGFβ1, ET-1, AngII, PDGF, OSM and IL-13 induceIL-11 secretion, and IL-11 also induces IL-11 expression in a positivefeedback loop. (FIG. 19B) Graph showing that the ELISA only detectsnative IL-11 secreted from cells, and does not detect recombinant IL-11used for the IL-11 stimulation condition. (FIG. 19C) and (FIG. 19D)Cells were stimulated with recombinant IL-11, IL-11 RNA was measured andthe native IL-11 protein level was measured in the cell culturesupernatant by ELISA at the indicated time points.

FIGS. 20A, 20B, 20C, 20D, 20E and 20F. Graphs and images showingmyofibroblast generation from, and production of ECM and cytokineexpression by, atrial fibroblasts in response to stimulation with TGFβ1or IL-11. (FIG. 20A) myofibroblast generation and ECM production byprimary atrial fibroblasts following stimulation with TGFβ1 or IL-11, asmeasured by fluorescence microscopy following staining for a α-SMA,collagen or periostin. (FIG. 20B) Collagen content of cell culturesupernatant as determined by Sirius Red staining. Secretion of thefibrosis markers (FIG. 20C) IL-6, (FIG. 20D) TIMP1 and (FIG. 20E) MMP2as measured by ELISA. (FIG. 20F) Activation of murine fibroblasts bystimulation with human or mouse recombinant IL-11. * P<0.05, ** P<0.01,*** P<0.001, **** P<0.0001 [Mean±SD, Dunnett].

FIGS. 21A, 21B and 21C. Graphs showing the profibrotic effect of IL-11.(FIG. 21A) Mouse fibroblasts from different tissues of origin can beactivated by IL-11 and display increased ECM production. [Mean±SD,Dunnett]. Injection of mice with recombinant IL-11 or Ang II results in(FIG. 21B) an increase in organ weight [Mean±SEM], and (FIG. 21C) anincrease in collagen content (as determined by HPA assay). * P<0.05, **P<0.01, *** P<0.001, **** P<0.0001 [Mean±SD, Dunnett].

FIGS. 22A, 22B, 22C, 22D, 22E and 22F. Graphs and images showing thatIL-11 is required the pro-fibrotic effects of TGFβ1 on fibroblasts.(FIG. 22A) myofibroblast generation and ECM production by primary atrialfibroblasts, with or without stimulation with TGFβ1, and in thepresence/absence of neutralising anti-IL-11 antibody or isotype controlIgG, as measured by fluorescence microscopy following staining for (FIG.22A) α-SMA, (FIG. 22B) EdU or (FIG. 22C) Periostin. (FIG. 22D to FIG.22F) Secretion of the fibrosis markers (FIG. 22D) IL-6, (FIG. 22E)TIMP1, and (FIG. 22F) MMP2 was analysed by ELISA. Fluorescence wasnormalized to the control group without stimulation. [Mean±SD,Dunnett] * P<0.05, ** P<0.01, *** P<0.001 or **** P<0.0001.

FIGS. 23A and 23B. Graphs and images showing the effect ofneutralisation of IL-11 on collagen production triggered by TGFβ1.Collagen production by cardiac fibroblasts with or without stimulationwith TGFβ1, and in the presence/absence of neutralising anti-IL-11antibody or isotype control IgG, as determined by (FIG. 23A) Operettaassay or (FIG. 23B) Sirius Red staining. [Mean±SD, Dunnett] * P<0.05, **P<0.01, *** P<0.001 or **** P<0.0001.

FIG. 24. Graphs showing the ability of various IL-11 and IL-11Rαantagonists to inhibit fibrosis. Human atrial fibroblasts were treatedwith neutralizing antibody against IL-11, neutralizing antibody againstIL-11Rα, decoy IL-11 receptor molecule that binds to IL-11, siRNA thatdownregulates IL-11 expression or siRNA that downregulates IL-11RAexpression and the effect on the TGFβ1-driven pro-fibrotic response infibroblasts in vitro was analysed. [Mean±SD, Dunnett] * P<0.05, **P<0.01, *** P<0.001 or **** P<0.0001.

FIGS. 25A, 25B, 25C and 25D. Bar charts showing the response offibroblasts from IL-11-RA knockout mice to pro-fibrotic treatment.Fibroblasts derived from IL-11RA WT (+/+), Heterozygous (+/−) andHomozygous null (−/−) mice were incubated for 24 h with TGFβ1, IL-11 orAngII (5 ng/ml). (FIG. 25A) Percentage of myofibroblasts as determinedby analysis αSMA content, (FIG. 25B) Percentage proliferating cells asdetermined by staining for EdU, (FIG. 25C) Collagen content and (FIG.25D) ECM production as measured by detection of periostin [Mean±SD].

FIGS. 26A and 26B. Graphs showing the effect of IL-11 neutralisation onfibrosis in response to various pro-fibrotic stimuli. Fibroblasts werecultured in vitro in the presence/absence of various differentpro-fibrotic factors, and in the presence/absence of neutralisinganti-IL-11 antibody or pan anti-TGFβ antibody (FIG. 26A) Collagenproduction and (FIG. 26B) myofibroblast generation as determined byanalysis of αSMA expression. [Mean±SD, Dunnett] * P<0.05, ** P<0.01, ***P<0.001 or **** P<0.0001.

FIGS. 27A, 27B, 27C and 27D. Bar charts showing expression of markers offibrosis in the atrium and heart of WT and IL-11RA (−/−) animalsfollowing treatment with AngII treatment. (FIG. 27A) Collagen content,as measured by hydroxyproline assay. (FIG. 27B) Collagen (Col1A2)expression. (FIG. 27C) αSMA (ACTA2) expression. (FIG. 27D) Fibronectin(Fn1) expression.

FIG. 28. Graphs showing the effect of IL-11RA knockout on folate-inducedkidney fibrosis as measured by collagen content in kidney tissue.

FIGS. 29A, 29B and 29C. Schematics of the experimental procedures foranalysing fibrosis in (FIG. 29A) lung, (FIG. 29B) skin and (FIG. 29C)eye for IL-11RA −/− mice as compared to IL-11RA +/+ mice.

FIGS. 30A and 30B. Scatterplots showing fold change in gene expression.(FIG. 30A) Fold changes in gene expression in fibroblasts followingstimulation with TGFβ1, IL-11 or TGFβ1 and IL-11. (FIG. 30B) Foldchanges in gene expression in fibroblasts obtained from IL-11RA+/+ andIL-11RA−/− mice following stimulation with TGFβ1.

FIGS. 31A and 31B. Photographs showing the effect of IL-11RA knockout onwound healing and fibrosis in the eye following trabeculectomy(filtration surgery). (FIG. 31A) Eye sections of IL-11RA+/+ (WT) andIL-11RA−/− (KO) animals 7 days after filtration surgery. (FIG. 31B)Maturation of collagen fibres as evaluated by picro-siriusred/polarization light technique (Szendroi et al. 1984, Acta MorpholHung 32, 47-55); more fibrosis is observed in WT mice than KO mice.

FIGS. 32A and 32B. Graphs showing the effect of decoy IL-11 receptors onfibrosis in response to stimulation with TGFβ1. Fibroblasts werecultured in vitro in the presence/absence of TGFβ1 (5 ng/ml), in thepresence or absence of (FIG. 32A) D11R1 (Decoy Receptor 50aa Linker) or(FIG. 32B) D11R2 (Decoy Receptor 33aa Linker), at various differentconcentrations. Myofibroblast generation after 24 hours (i.e. thepercentage of activated fibroblasts) was determined by analysis of αSMAexpression.

FIG. 33. Table showing SNPs regulation of IL-11 VST_(stim) in trans.

FIG. 34. Table showing SNPs regulation of IL-11 VST_(stim)−VST_(unstim)in cis.

FIG. 35. Table showing SNPs regulation of IL-11 VST_(stim)−VST_(unstim)in trans.

FIGS. 36A, 36B, 36C and 36D. Charts showing regulation of IL-11 responseby local SNPs (FIGS. 36A, 36B, 36C and 36D). The RNA of unstimulated andstimulated (TGFB1, 5 ng/ml, 24 h) fibroblasts derived from 69 genotypedindividuals was sequenced. Samples were grouped according to genotypeand the increase in IL-11 expression (VST_(stim)−VST_(unstim)) wascompared between groups with 0, 1 or 2 minor alleles.

FIG. 37. Charts showing regulation of IL-11 response by distant SNPs.The RNA of unstimulated and stimulated (TGFB1, 5 ng/ml, 24 h)fibroblasts derived from 69 genotyped individuals was sequenced. Sampleswere grouped according to genotype and the increase in IL11 expression(VST_(stim)−VST_(unstim)) was compared between groups with 0, or 1 minorallele.

FIGS. 38A, 38B, 38C and 38D. Graphs showing that IL-11 is required thepro-fibrotic effects of TGFβ1 in liver fibroblasts. Activation andproliferation of primary human liver fibroblasts, with or withoutstimulation with TGFβ1, and in the presence/absence of neutralisinganti-IL-11 antibody or isotype control IgG, as measured by analysis ofthe proportion of (FIG. 38A) α-SMA positive cells, (FIG. 38B) EdUpositive cells, (FIG. 38C) Collagen positive cells and (FIG. 38D)Periostin positive cells as compared to the unstimulated cells(Baseline). [Mean±SD, Dunnett] * P<0.05, ** P<0.01, *** P<0.001 or ****P<0.0001.

FIG. 39. Bar chart showing that IL-11 is required for the pro-fibroticeffects of TGFβ1 in skin fibroblasts. Activation of mouse skinfibroblasts, with or without stimulation with TGFβ1, and in thepresence/absence of neutralising anti-IL-11 antibody, as measured byanalysis of the percentage of α-SMA positive cells (activatedfibroblasts).

FIG. 40. Bar chart showing lung fibroblast cell migration with andwithout IL-11 signaling. Migration of lung fibroblasts from IL-11RA+/+(WT) and IL-11RA−/− (KO) animals was analysed in an in vitro scratchassay without stimulus, or in the presence of TGFβ1 or IL-11.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

IL-11 and IL-11 Receptor

Interleukin 11 (IL-11), also known as adipogenesis inhibitory factor, isa pleiotropic cytokine and a member of the IL-6 family of cytokines thatincludes IL-6, IL-11, IL-27, IL-31, oncostatin, leukemia inhibitoryfactor (LIF), cardiotrophin-1 (CT-1), cardiotrophin-like cytokine (CLC),ciliary neurotrophic factor (CNTF) and neuropoetin (NP-1).

IL-11 is transcribed with a canonical signal peptide that ensuresefficient secretion from cells. The immature form of human IL-11 is a199 amino acid polypeptide whereas the mature form of IL-11 encodes aprotein of 178 amino acid residues (Garbers and Scheller., Biol. Chem.2013; 394(9):1145-1161). The human IL-11 amino acid sequence isavailable under UniProt accession no. P20809 (P20809.1 GI:124294).Recombinant human IL-11 (oprelvekin) is also commercially available.IL-11 from other species, including mouse, rat, pig, cow, severalspecies of bony fish and primates, have also been cloned and sequenced.

In this specification IL-11 refers to an IL-11 from any species andincludes isoforms, fragments, variants or homologues of an IL-11 fromany species. In preferred embodiments the species is human (Homosapiens). Isoforms, fragments, variants or homologues of an IL-11 mayoptionally be characterised as having at least 70%, preferably one of80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% aminoacid sequence identity to the amino acid sequence of immature or matureIL-11 from a given species, e.g. human. Isoforms, fragments, variants orhomologues of an IL-11 may optionally be characterised by ability tobind IL-11Rα (preferably from the same species) and stimulate signaltransduction in cells expressing IL-11Rα and gp130 (e.g. as described inCurtis et al. Blood, 1997, 90(11); or Karpovich et al. Mol. Hum. Reprod.2003 9(2): 75-80). A fragment of IL-11 may be of any length (by numberof amino acids), although may optionally be at least 25% of the lengthof mature IL-11 and may have a maximum length of one of 50%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the lengthof mature IL-11. A fragment of IL-11 may have a minimum length of 10amino acids, and a maximum length of one of 15, 20, 25, 30, 40, 50, 100,110, 120, 130, 140, 150, 160, 170, 180, 190 or 195 amino acids

IL-11 signals through a homodimer of the ubiquitously expressedβ-receptor glycoprotein 130 (gp130; also known as glycoprotein 130,IL6ST, IL6-beta or CD130). Gp130 is a transmembrane protein that formsone subunit of the type I cytokine receptor with the IL-6 receptorfamily. Specificity is gained through an individual IL-11 α-receptor(IL-11Rα), which does not directly participate in signal transduction,although the initial cytokine binding event to the α-receptor leads tothe final complex formation with the β-receptors. IL-11 activates adownstream signaling pathway, which is predominantly themitogen-activated protein kinase (MAPK)-cascade and the Januskinase/signal transducer and activator of transcription (Jak/STAT)pathway (Garbers and Scheller, supra).

Human gp130 (including the 22 amino acid signal peptide) is a 918 aminoacid protein, and the mature form is 866 amino acids, comprising a 597amino acid extracellular domain, a 22 amino acid transmembrane domain,and a 277 amino acid intracellular domain. The extracellular domain ofthe protein comprises the cytokine-binding module (CBM) of gp130. TheCBM of gp130 comprises the Ig-like domain D1, and the fibronectin-typeIII domains D2 and D3 of gp130. The amino acid sequence of human gp130is available from Genbank accession no. NP_002175.2.

Human IL-11Rα is a 422 amino acid polypeptide (Genbank accession no.NP_001136256.1 GI:218505839) and shares ˜85% nucleotide and amino acidsequence identity with the murine IL-11Rα (Du and Williams., Blood Vol,89, No, 11, Jun. 1, 1997). Two isoforms of IL-11Rα have been reported,which differ in the cytoplasmic domain (Du and Williams, supra). TheIL-11 receptor α-chain (IL-11Rα) shares many structural and functionalsimilarities with the IL-6 receptor α-chain (IL-6Rα). The extracellulardomain shows 24% amino acid identity including the characteristicconserved Trp-Ser-X-Trp-Ser (WSXWS) motif. The short cytoplasmic domain(34 amino acids) lacks the Box 1 and 2 regions that are required foractivation of the JAK/STAT signaling pathway.

IL-11Rα binds its ligand with a low affinity (Kd˜10 nmol/L) and alone isinsufficient to transduce a biological signal. The generation of a highaffinity receptor (Kd˜400 to 800 pmol/L) capable of signal transductionrequires co-expression of the IL-11Rα and gp130 (Curtis et al (Blood1997 Dec. 1; 90 (11):4403-12; Hilton et al., EMBO J 13:4765, 1994;Nandurkar et al., Oncogene 12:585, 1996). Binding of IL-11 tocell-surface IL-11Rα induces heterodimerization, tyrosinephosphorylation, activation of gp130 and MAPK and/or Jak/STAT signalingas described above.

The receptor binding sites on murine IL-11 have been mapped and threesites—sites I, II and III—identified. Binding to gp130 is reduced bysubstitutions in the site II region and by substitutions in the site IIIregion. Site III mutants show no detectable agonist activity and haveIL-11Rα antagonist activity (Cytokine Inhibitors Chapter 8; edited byGennaro Ciliberto and Rocco Savino, Marcel Dekker, Inc. 2001).

In principle, a soluble IL-11Rα can also form biologically activesoluble complexes with IL-11 (Pflanz et al., 1999 FEBS Lett, 450,117-122) raising the possibility that, similar to IL-6, IL-11 may insome instances bind soluble IL-11Rα prior to binding cell-surface gp130(Garbers and Scheller, supra). Curtis et al (Blood 1997 Dec. 1; 90(11):4403-12) describe expression of a soluble murine IL-11 receptoralpha chain (sIL-11R) and examined signaling in cells expressing gp130.In the presence of gp130 but not transmembrane IL-11R the sIL-11Rmediated IL-11 dependent differentiation of M1 leukemic cells andproliferation in Ba/F3 cells and early intracellular events includingphosphorylation of gp130, STAT3 and SHP2 similar to signaling throughtransmembrane IL-11R.

In this specification an IL-11 receptor (IL-11R) refers to a polypeptidecapable of binding IL-11 and inducing signal transduction in cellsexpressing gp130. An IL-11 receptor may be from any species and includesisoforms, fragments, variants or homologues of an IL-11 receptor fromany species. In preferred embodiments the species is human (Homosapiens). In some embodiments the IL-11 receptor may be IL-11Rα.Isoforms, fragments, variants or homologues of an IL-11Rα may optionallybe characterised as having at least 70%, preferably one of 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acidsequence identity to the amino acid sequence of IL-11Rα from a givenspecies, e.g. human. Isoforms, fragments, variants or homologues of anIL-11Rα may optionally be characterised by ability to bind IL-11(preferably from the same species) and stimulate signal transduction incells expressing the IL-11Rα and gp130 (e.g. as described in Curtis etal. Blood, 1997, 90(11) or Karpovich et al. Mol. Hum. Reprod. 2003 9(2):75-80). A fragment of an IL-11 receptor may be of any length (by numberof amino acids), although may optionally be at least 25% of the lengthof the mature IL-11Rα and have a maximum length of one of 50%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the lengthof the mature IL-11Rα. A fragment of an IL-11 receptor fragment may havea minimum length of 10 amino acids, and a maximum length of one of 15,20, 25, 30, 40, 50, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 250, 300, 400, or 415 amino acids.

Agent Capable of Inhibiting the Action of IL-11

The IL-11 signaling pathway offers multiple routes for inhibition ofIL-11 signaling. For example, inhibition may be achieved by preventingor reducing the binding of IL-11 to an IL-11 receptor. As a result,suitable agents may target either IL-11 or its receptor.

In some embodiments agents capable of inhibiting the action of IL-11 maybind to IL-11 and prevent or reduce IL-11 mediated signalling, e.g.through an IL-11 receptor. In some embodiments agents capable ofinhibiting the action of IL-11 may bind to the IL-11 receptor andprevent or reduce IL-11 stimulated signalling.

Agents that bind to IL-11 may inhibit IL-11 mediated signalling byblocking the binding of IL-11 to an IL-11 receptor and/or by reducingthe amount of IL-11 available to bind to its receptor. Suitable IL-11binding agents may be IL-11 inhibitors or IL-11 antagonists.

IL-11 binding agents, e.g. anti-IL-11 antibodies, according to thepresent invention may exhibit at least one of the following properties:

-   -   a) Bind to human IL-11 with a K_(D) of 1 μM or less, preferably        one of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM or ≤100 pM;    -   b) Inhibit IL-11 mediated signaling via the IL-11Rα receptor,        e.g. in a cell based assay in which the cells co-express IL-11Rα        and gp130. Suitable cell based assays are ³H-thymidine        incorporation and Ba/F3 cell proliferation assays described in        e.g. Curtis et al. Blood, 1997, 90(11) and Karpovich et al. Mol.        Hum. Reprod. 2003 9(2): 75-80. For example, IC₅₀ for an IL-11        binding agent may be determined by culturing Ba/F3 cells        expressing IL-11Rα and gp130 in the presence of human IL-11 and        the IL-11 binding agent, and measuring ³H-thymidine        incorporation into DNA. Suitable IL-11 binding agents may        exhibit an IC₅₀ of 10 μg/ml or less, preferably one of ≤5 μg/ml,        ≤4 μg/ml, ≤3.5 μg/ml, ≤3 μg/ml, ≤2 μg/ml, ≤1 μg/ml, ≤0.9 μg/ml,        ≤0.8 μg/ml, ≤0.7 μg/ml, ≤0.6 μg/ml, or ≤0.5 μg/ml in such an        assay.    -   c) Inhibit fibroblast proliferation, e.g. proliferation of        cardiac/atrial fibroblasts. This can, for example, be evaluated        in an assay wherein fibroblasts are stimulated with IL-11 or        TGFβ1 and cell proliferation is monitored as described herein.    -   d) Inhibit myofibroblast generation, e.g. from cardiac/atrial        fibroblasts. This can, for example, be evaluated in an assay        wherein fibroblasts are stimulated with IL-11 or TGFβ1 and        myofibroblast generation is monitored, e.g. by measuring αSMA        levels.    -   e) Inhibit extracellular matrix production by fibroblasts, e.g.        cardiac/atrial fibroblasts. This can, for example, be evaluated        in an assay wherein fibroblasts are stimulated with IL-11 or        TGFβ1 and production of extracellular matrix components is        measured.    -   f) Inhibit collagen and/or periostin gene or protein expression        in fibroblasts, e.g. cardiac/atrial fibroblasts. This can, for        example, be evaluated in an assay wherein fibroblasts are        stimulated with IL-11 or TGFβ1 and collagen and/or periostin        gene or protein expression is measured.

IL-11 binding agents may be of any kind, but in some embodiments anIL-11 binding agent may be an antibody, polypeptide, peptide,oligonucleotide, aptamer or small molecule.

Suitable anti-IL-11 antibodies will preferably bind to IL-11 (theantigen), preferably human IL-11, and may have a dissociation constant(K_(D)) of one of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM or ≤100 pM. Bindingaffinity of an antibody for its target is often described in terms ofits dissociation constant (K_(D)). Binding affinity can be measured bymethods known in the art, such as by Surface Plasmon Resonance (SPR), orby a radiolabeled antigen binding assay (RIA) performed with the Fabversion of the antibody and antigen molecule.

Anti-IL-11 antibodies may be antagonist antibodies that inhibit orreduce a biological activity of IL-11.

Anti-IL-11 antibodies may be neutralising antibodies that neutralise thebiological effect of IL-11, e.g. its ability to stimulate productivesignaling via an IL-11 receptor.

Neutralising activity may be measured by ability to neutralise IL-11induced proliferation in the T11 mouse plasmacytoma cell line (Nordan,R. P. et al. (1987) J. Immunol. 139:813).

Examples of known anti-IL-11 antibodies include monoclonal antibodyclone 6D9A, clone KT8 (Abbiotec), clone M3103F11 (BioLegend), clone 1F1,clone 3C6 (Abnova Corporation), clone GF1 (LifeSpan Biosciences), clone13455 (Source BioScience) and clone 22626 (R & D Systems, used inBockhorn et al. Nat. Commun. (2013) 4(0):1393; Monoclonal MouseIgG_(2A); Catalog No. MAB218; R&D Systems, MN, USA).

Antibodies may optionally be selected to exhibit substantially nocross-reactivity with one or more of human, e.g. recombinant human,IL-6, CNTF, LIF, OSM, CLC or CT-1.

Peptide or polypeptide based IL-11 binding agents may be based on theIL-11 receptor, e.g. a IL-11 binding fragment of an IL-11 receptor. Inone embodiment, suitable IL-11 binding agents may comprise an IL-11binding fragment of the IL-11Rα chain, and may preferably be solubleand/or exclude one or more, or all, of the transmembrane domain(s). Suchmolecules may be described as decoy receptors.

Curtis et al (Blood 1997 Dec. 1; 90 (11):4403-12) report that a solublemurine IL-11 receptor alpha chain (sIL-11R) was capable of antagonizingthe activity of IL-11 when tested on cells expressing the transmembraneIL-11R and gp130. They proposed that the observed IL-11 antagonism bythe sIL-11R depends on limiting numbers of gp130 molecules on cellsalready expressing the transmembrane IL-11R.

The use of soluble decoy receptors as the basis for inhibition of signaltransduction and therapeutic intervention has also been reported forother signaling molecule:receptor pairs, e.g. VEGF and the VEGF receptor(De-Chao Yu et al., Molecular Therapy (2012); 20 5, 938-947; Konner andDupont Clin Colorectal Cancer 2004 October; 4 Suppl 2:S81-5).

As such, in some embodiments an IL-11 binding agent may be provided inthe form of a decoy receptor, e.g. a soluble IL-11 receptor. Competitionfor IL-11 provided by a decoy receptor has been reported to lead toIL-11 antagonist action (Curtis et al., supra).

Decoy IL-11 receptors preferably bind IL-11 and/or IL-11 containingcomplexes, and thereby make these species unavailable for binding togp130, IL-11Rα and/or gp130:IL-11Rα receptors. As such, they act as‘decoy’ receptors for IL-11 and IL-11 containing complexes, much in thesame way that etanercept acts as a decoy receptor for TNFα. IL-11mediated signaling is reduced as compared to the level of signaling inthe absence of the decoy receptor.

Decoy IL-11 receptors preferably bind to IL-11 through one or morecytokine binding modules (CBMs). The CBMs are, or are derived from orhomologous to, the CBMs of naturally occurring receptor molecules forIL-11. For example, decoy IL-11 receptors may comprise, or consist of,one or more CBMs which are from, are derived from or homologous to theCBM of gp130 and/or IL-11Rα.

In some embodiments, a decoy IL-11 receptor may comprise, or consist of,an amino acid sequence corresponding to the cytokine binding module ofgp130. In some embodiments, a decoy IL-11 receptor may comprise an aminoacid sequence corresponding to the cytokine binding module of IL-11Rα.Herein, an amino acid sequence which ‘corresponds’ to a reference regionor sequence of a given peptide/polypeptide has at least 60%, e.g. one ofat least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% sequence identity to the amino acid sequence of thereference region/sequence. The gp130, IL-11Rα and IL-11 may be from anyspecies, and include isoforms, fragments, variants or homologues fromany species.

In some embodiments a decoy receptor may be able to bind IL-11, e.g.with binding affinity of at least 100 μM or less, optionally one of 10μM or less, 1 μM or less, 100 nM or less, or about 1 to 100 nM. In someembodiments a decoy receptor may comprise all or part of the IL-11binding domain and may optionally lack all or part of the transmembranedomains. The decoy receptor may optionally be fused to an immunoglobulinconstant region, e.g. IgG Fc region.

In some embodiments an IL-11 binding agent may be provided in the formof a small molecule inhibitor of IL-11, e.g. IL-11 inhibitor describedin Lay et al., Int. J. Oncol. (2012); 41(2): 759-764.

Agents that bind to an IL-11 receptor (IL-11R) may inhibit IL-11mediated signalling by blocking the binding of IL-11 to an IL-11R or bypreventing signal transduction via the gp130 co-receptors. SuitableIL-11R binding agents may be IL-11R inhibitors or IL-11R antagonists. Inpreferred embodiments the IL-11R is IL-11Rα and suitable binding agentsmay bind the IL-11Rα polypeptide and may be inhibitors or antagonists ofIL-11Rα.

IL-11R binding agents, e.g. anti-IL-11R antibodies, according to thepresent invention may exhibit at least one of the following properties:

-   -   (a) Bind to human IL-11R with a K_(D) of 1 μM or less,        preferably one of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM or ≤100 pM;    -   (b) Inhibit IL-11R signaling, e.g. in a cell based assay in        which the cells co-express IL-11Rα and gp130. Suitable cell        based assays are ³H-thymidine incorporation and Ba/F3 cell        proliferation assays described in e.g. Curtis et al. Blood,        1997, 90(11) and Karpovich et al. Mol. Hum. Reprod. 2003 9(2):        75-80. For example, IC₅₀ for an IL-11R binding agent may be        determined by culturing Ba/F3 cells expressing IL-11Rα and gp130        in the presence of human IL-11 and the IL-11R binding agent, and        measuring ³H-thymidine incorporation into DNA. Suitable IL-11R        binding agents may exhibit an IC₅₀ of 10 μg/ml or less,        preferably one of ≤5 μg/ml, ≤4 μg/ml, ≤3.5 μg/ml, ≤3 μg/ml, ≤2        μg/ml, ≤1 μg/ml, ≤0.9 μg/ml, ≤0.8 μg/ml, ≤0.7 μg/ml, ≤0.6 μg/ml,        or ≤0.5 μg/ml in such an assay.    -   (c) Inhibit fibroblast proliferation, e.g. proliferation of        cardiac/atrial fibroblasts. This can, for example, be evaluated        in an assay wherein fibroblasts are stimulated with IL-11 or        TGFβ1 and cell proliferation is monitored as described herein.    -   (d) Inhibit myofibroblast generation, e.g. from cardiac/atrial        fibroblasts. This can, for example, be evaluated in an assay        wherein fibroblasts are stimulated with IL-11 or TGFβ1 and        myofibroblast generation is monitored, e.g. by measuring αSMA        levels.    -   (e) Inhibit extracellular matrix production by fibroblasts, e.g.        cardiac/atrial fibroblasts. This can, for example, be evaluated        in an assay wherein fibroblasts are stimulated with IL-11 or        TGFβ1 and production of extracellular matrix components is        measured.    -   (f) Inhibit collagen and/or periostin gene or protein expression        in fibroblasts, e.g. cardiac/atrial fibroblasts. This can, for        example, be evaluated in an assay wherein fibroblasts are        stimulated with IL-11 or TGFβ1 and collagen and/or periostin        gene or protein expression is measured.

IL-11R binding agents may be of any kind, but in some embodiments anIL-11R binding agent may be an antibody, polypeptide, peptide,oligonucleotide, aptamer or small molecule.

Suitable anti-IL-11R antibodies will preferably bind to IL-11R (theantigen), preferably human IL-11R, and may have a dissociation constant(K_(D)) of one of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM or ≤100 pM. Bindingaffinity of an antibody for its target is often described in terms ofits dissociation constant (K_(D)). Binding affinity can be measured bymethods known in the art, such as by Surface Plasmon Resonance (SPR), orby a radiolabeled antigen binding assay (RIA) performed with the Fabversion of the antibody and antigen molecule.

Anti-IL-11R antibodies may be antagonist antibodies that inhibit orreduce a biological activity of IL-11R. Anti-IL-11R antibodies may beantagonist antibodies that inhibit or reduce any function of IL-11R, inparticular signaling. For example, antagonist IL-11R antibodies mayinhibit or prevent binding of IL-11 to IL-11R, or may inhibit or preventassociation of IL-11Rα with gp130 to form a functional receptor complexcapable of productive signaling, e.g. in response to IL-11 binding.

Anti-IL-11R antibodies may be neutralising antibodies that neutralisethe biological effect of IL-11R, e.g. its ability to initiate productivesignaling mediated by binding of IL-11.

Neutralising activity may be measured by ability to neutralise IL-11induced proliferation in the T11 mouse plasmacytoma cell line (Nordan,R. P. et al. (1987) J. Immunol. 139:813).

Examples of known anti-IL-11R antibodies include monoclonal antibodyclone 025 (Sino Biological), clone EPR5446 (Abcam), clone 473143 (R & DSystems), clones 8E2 and 8E4 described in US 2014/0219919 A1 and themonoclonal antibodies described in Blanc et al (J. Immunol Methods. 2000Jul. 31; 241(1-2);43-59).

Peptide or polypeptide based IL-11R binding agents may be based onIL-11, e.g. mutant, variant or binding fragment of IL-11. Suitablepeptide or polypeptide based agents may bind to IL-11R in a manner thatdoes not lead to initiation of signal transduction or producessub-optimal signaling. IL-11 mutants of this kind may act as competitiveinhibitors of endogenous IL-11.

For example, W147A is an IL-11 antagonist in which the amino acid 147 ismutated from a tryptophan to an alanine, which destroys the so-called‘site III’ of IL-11. This mutant can bind to the IL-11R, but engagementof the gp130 homodimer fails, resulting in efficient blockade of IL-11signaling (Underhill-Day et al., 2003; Endocrinology 2003 August;144(8):3406-14). Lee et al (Am J respire Cell Mol Biol. 2008 December;39(6):739-746) also report the generation of an IL-11 antagonist mutant(a “mutein”) capable of specifically inhibiting the binding of IL-11 toIL-11Rα.

Menkhorst et al (Biology of Reproduction May 1, 2009 vol. 80 no. 5920-927) describe a PEGylated IL-11 antagonist, PEGIL11A (CSL Limited,Parkvill, Victoria, Australia) which is effective to inhibit IL-11action in female mice.

Pasqualini et al. Cancer (2015) 121(14):2411-2421 describe aligand-directed, peptidomimetic drug, bone metastasis-targetingpeptidomimetic-11 (BMTP-11) capable of binding to IL-11Rα.

In some embodiments an IL-11R binding agent may be provided in the formof a small molecule inhibitor of IL-11R.

The inventors have identified that upregulation of IL-11 expression isconsistent with the molecular mechanism of fibrosis and that inhibitionof IL-11 activity leads to a reduction in the molecular basis forfibrosis. Accordingly, in some aspects of the present inventiontreatment, prevention or alleviation of fibrosis may be provided byadministration of an agent capable of preventing or reducing theexpression of IL-11 by cells of the subject, e.g. by fibroblasts ormyofibroblasts.

Suitable agents may be of any kind, but in some embodiments an agentcapable of preventing or reducing the expression of IL-11 may be a smallmolecule or an oligonucleotide.

Taki et al (Clin Exp Immunol. 1998 April; 112(1): 133-138) report areduction in the expression of IL-11 in rheumatoid synovial cells upontreatment with indomethacin, dexamethasone or interferon-gamma (IFNγ).

In some embodiments an agent capable of preventing or reducing theexpression of IL-11 may be an oligonucleotide capable of repressing orsilencing expression of IL-11.

Accordingly, the present invention also includes the use of techniquesknown in the art for the therapeutic down regulation of IL-11expression. These include the use of antisense oligonucleotides and RNAinterference (RNAi). As in other aspects of the present invention, thesetechniques may be used in the treatment of fibrosis.

Accordingly, in one aspect of the present invention a method of treatingor preventing fibrosis is provided, the method comprising administeringto a subject in need of treatment a therapeutically effective amount ofan agent capable of preventing or reducing the expression of IL-11,wherein the agent comprises a vector comprising a therapeuticoligonucleotide capable of repressing or silencing expression of IL-11.

In another aspect of the present invention a method of treating orpreventing fibrosis is provided, the method comprising administering toa subject in need of treatment a therapeutically effective amount of anagent capable of preventing or reducing the expression of IL-11, whereinthe agent comprises an oligonucleotide vector, optionally a viralvector, encoding a therapeutic oligonucleotide capable of beingexpressed in cells of the subject, the expressed therapeuticoligonucleotide being capable of repressing or silencing expression ofIL-11.

The ability of an agent to prevent or reduce the expression of IL-11 maybe assayed by determining the ability of the agent to inhibit IL-11 geneor protein expression by fibroblasts or myofibroblasts, e.g.cardiac/atrial fibroblasts or myofibroblasts. This can, for example, beevaluated in an assay wherein fibroblasts or myofibroblasts arestimulated with IL-11 or TGFβ1, and IL-11 gene or protein expression ismeasured.

Reducing the amount of IL-11R available for binding to IL-11 andinitiation of productive signaling provides an alternative means ofreducing the level of IL-11 stimulated signaling. Accordingly, inrelated aspects of the present invention, treatment, prevention oralleviation of fibrosis may be provided by administration of an agentcapable of preventing or reducing the expression of IL-11R by cells ofthe subject, e.g. by fibroblasts or myofibroblasts.

In some embodiments an agent capable of preventing or reducing theexpression of IL-11R may be an oligonucleotide capable of repressing orsilencing expression of IL-11R.

Accordingly, the present invention also includes the use of techniquesknown in the art for the therapeutic down regulation of IL-11Rexpression. These include the use of antisense oligonucleotides and RNAinterference (RNAi). As in other aspects of the present invention, thesetechniques may be used in the treatment of fibrosis.

Accordingly, in one aspect of the present invention a method of treatingor preventing fibrosis is provided, the method comprising administeringto a subject in need of treatment a therapeutically effective amount ofan agent capable of preventing or reducing the expression of IL-11R,wherein the agent comprises a vector comprising a therapeuticoligonucleotide capable of repressing or silencing expression of IL-11R.

In another aspect of the present invention a method of treating orpreventing fibrosis is provided, the method comprising administering toa subject in need of treatment a therapeutically effective amount of anagent capable of preventing or reducing the expression of IL-11R,wherein the agent comprises an oligonucleotide vector, optionally aviral vector, encoding a therapeutic oligonucleotide capable of beingexpressed in cells of the subject, the expressed therapeuticoligonucleotide being capable of repressing or silencing expression ofIL-11R.

The ability of an agent to prevent or reduce the expression of IL-11Rmay be assayed by determining the ability of the agent to inhibit IL-11Rgene or protein expression by fibroblasts or myofibroblasts, e.g.cardiac/atrial fibroblasts or myofibroblasts. This can, for example, beevaluated in an assay wherein fibroblasts or myofibroblasts arestimulated with IL-11 or TGFβ1, and IL-11R gene or protein expression ismeasured.

In preferred embodiments, the IL-11R may be IL-11Rα.

Antibodies

In this specification “antibody” includes a fragment or derivative of anantibody, or a synthetic antibody or synthetic antibody fragment.

Antibodies may be provided in isolated or purified form. Antibodies maybe formulated as a pharmaceutical composition or medicament.

In view of today's techniques in relation to monoclonal antibodytechnology, antibodies can be prepared to most antigens. Theantigen-binding portion may be a part of an antibody (for example a Fabfragment) or a synthetic antibody fragment (for example a single chainFv fragment [ScFv]). Suitable monoclonal antibodies to selected antigensmay be prepared by known techniques, for example those disclosed in“Monoclonal Antibodies: A manual of techniques”, H Zola (CRC Press,1988) and in “Monoclonal Hybridoma Antibodies: Techniques andApplications”, J G R Hurrell (CRC Press, 1982). Chimaeric antibodies arediscussed by Neuberger et al (1988, 8th International BiotechnologySymposium Part 2, 792-799).

Monoclonal antibodies (mAbs) are useful in the methods of the inventionand are a homogenous population of antibodies specifically targeting asingle epitope on an antigen.

Polyclonal antibodies are useful in the methods of the invention.Monospecific polyclonal antibodies are preferred. Suitable polyclonalantibodies can be prepared using methods well known in the art.

Antigen binding fragments of antibodies, such as Fab and Fab₂ fragmentsmay also be used/provided as can genetically engineered antibodies andantibody fragments. The variable heavy (V_(H)) and variable light(V_(L)) domains of the antibody are involved in antigen recognition, afact first recognised by early protease digestion experiments. Furtherconfirmation was found by “humanisation” of rodent antibodies. Variabledomains of rodent origin may be fused to constant domains of humanorigin such that the resultant antibody retains the antigenicspecificity of the rodent parented antibody (Morrison et al (1984) Proc.Natl. Acad. Sd. USA 81, 6851-6855).

That antigenic specificity is conferred by variable domains and isindependent of the constant domains is known from experiments involvingthe bacterial expression of antibody fragments, all containing one ormore variable domains. These molecules include Fab-like molecules(Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al(1988) Science 240, 1038); single-chain Fv (ScFv) molecules where theV_(H) and V_(L) partner domains are linked via a flexible oligopeptide(Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl.Acad. Sd. USA 85, 5879) and single domain antibodies (dAbs) comprisingisolated V domains (Ward et al (1989) Nature 341, 544). A general reviewof the techniques involved in the synthesis of antibody fragments whichretain their specific binding sites is to be found in Winter & Milstein(1991) Nature 349, 293-299.

By “ScFv molecules” we mean molecules wherein the V_(H) and V_(L)partner domains are covalently linked, e.g. by a flexible oligopeptide.

Fab, Fv, ScFv and dAb antibody fragments can all be expressed in andsecreted from E. coli, thus allowing the facile production of largeamounts of the said fragments.

Whole antibodies, and F(ab′)₂ fragments are “bivalent”. By “bivalent” wemean that the said antibodies and F(ab′)₂ fragments have two antigencombining sites. In contrast, Fab, Fv, ScFv and dAb fragments aremonovalent, having only one antigen combining site. Synthetic antibodieswhich bind to IL-11 or IL-11R may also be made using phage displaytechnology as is well known in the art.

Antibodies may be produced by a process of affinity maturation in whicha modified antibody is generated that has an improvement in the affinityof the antibody for antigen, compared to an unmodified parent antibody.Affinity-matured antibodies may be produced by procedures known in theart, e.g., Marks et al., Rio/Technology 10:779-783 (1992); Barbas et al.Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995);Jackson et al., J. Immunol. 154(7):331 0-15 9 (1995); and Hawkins et al,J. Mol. Biol. 226:889-896 (1992).

Antibodies according to the present invention preferably exhibitspecific binding to IL-11 or IL-11R. An antibody that specifically bindsto a target molecule preferably binds the target with greater affinity,and/or with greater duration than it binds to other targets. In oneembodiment, the extent of binding of an antibody to an unrelated targetis less than about 10% of the binding of the antibody to the target asmeasured, e.g., by ELISA, or by a radioimmunoassay (RIA). Alternatively,the binding specificity may be reflected in terms of binding affinitywhere the antibody binds to IL-11 or IL-11R with a K_(D) that is atleast 0.1 order of magnitude (i.e. 0.1×10^(n), where n is an integerrepresenting the order of magnitude) greater than the K_(D) of theantibody towards another target molecule, e.g. another member of theIL-11 family such as IL-6 or the IL-6 receptor. This may optionally beone of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or2.0.

Antibodies may be detectably labelled or, at least, capable ofdetection. Such antibodies being useful for both in vivo (e.g. imagingmethods) and in vitro (e.g. assay methods) applications For example, theantibody may be labelled with a radioactive atom or a coloured moleculeor a fluorescent molecule or a molecule which can be readily detected inany other way. Suitable detectable molecules include fluorescentproteins, luciferase, enzyme substrates, and radiolabels. The bindingmoiety may be directly labelled with a detectable label or it may beindirectly labelled. For example, the binding moiety may be anunlabelled antibody which can be detected by another antibody which isitself labelled. Alternatively, the second antibody may have bound to itbiotin and binding of labelled streptavidin to the biotin is used toindirectly label the first antibody.

Aspects of the present invention include bi-specific antibodies, e.g.composed of two different fragments of two different antibodies, suchthat the bi-specific antibody binds two types of antigen. One of theantigens is IL-11 or IL-11R, the bi-specific antibody comprising afragment as described herein that binds to IL-11 or IL-11R. The antibodymay contain a different fragment having affinity for a second antigen,which may be any desired antigen. Techniques for the preparation ofbi-specific antibodies are well known in the art, e.g. see Mueller, D etal., (2010 Biodrugs 24 (2): 89-98), Wozniak-Knopp G et al., (2010Protein Eng Des 23 (4): 289-297. Baeuerle, P A et al., (2009 Cancer Res69 (12): 4941-4944).

In some embodiments, the bispecific antibody is provided as a fusionprotein of two single-chain variable fragments (scFV) format, comprisinga V_(H) and V_(L) of a IL-11 or IL-11R binding antibody or antibodyfragment, and a V_(H) and V_(L) of an another antibody or antibodyfragment.

Bispecific antibodies and bispecific antigen binding fragments may beprovided in any suitable format, such as those formats described inKontermann MAbs 2012, 4(2): 182-197, which is hereby incorporated byreference in its entirety. For example, a bispecific antibody orbispecific antigen binding fragment may be a bispecific antibodyconjugate (e.g. an IgG2, F(ab′)₂ or CovX-Body), a bispecific IgG orIgG-like molecule (e.g. an IgG, scFv₄-Ig, IgG-scFv, scFv-IgG, DVD-Ig,IgG-sVD, sVD-IgG, 2 in 1-IgG, mAb², or Tandemab common LC), anasymmetric bispecific IgG or IgG-like molecule (e.g. a kih IgG, kih IgGcommon LC, CrossMab, kih IgG-scFab, mAb-Fv, charge pair or SEED-body), asmall bispecific antibody molecule (e.g. a Diabody (Db), dsDb, DART,scDb, tandAbs, tandem scFv (taFv), tandem dAb/VHH, triple body, triplehead, Fab-scFv, or F(ab′)₂-scFv₂), a bispecific Fc and C_(H)3 fusionprotein (e.g. a taFv-Fc, Di-diabody, scDb-C_(H)3, scFv-Fc-scFv,HCAb-VHH, scFv-kih-Fc, or scFv-kih-C_(H)3), or a bispecific fusionprotein (e.g. a scFv2-albumin, scDb-albumin, taFv-toxin, DNL-Fab₃,DNL-Fab₄-IgG, DNL-Fab₄-IgG-cytokine₂). See in particular FIG. 2 ofKontermann MAbs 2012, 4(2): 182-19.

Methods for producing bispecific antibodies include chemicallycrosslinking antibodies or antibody fragments, e.g. with reducibledisulphide or non-reducible thioether bonds, for example as described inSegal and Bast, 2001. Production of Bispecific Antibodies. CurrentProtocols in Immunology. 14:IV:2.13:2.13.1-2.13.16, which is herebyincorporated by reference in its entirety. For example,N-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) can be used tochemically crosslink e.g. Fab fragments via hinge region SH— groups, tocreate disulfide-linked bispecific F(ab)₂ heterodimers.

Other methods for producing bispecific antibodies include fusingantibody-producing hybridomas e.g. with polyethylene glycol, to producea quadroma cell capable of secreting bispecific antibody, for example asdescribed in D. M. and Bast, B. J. 2001. Production of BispecificAntibodies. Current Protocols in Immunology. 14:IV:2.13:2.13.1-2.13.16.

Bispecific antibodies and bispecific antigen binding fragments can alsobe produced recombinantly, by expression from e.g. a nucleic acidconstruct encoding polypeptides for the antigen binding molecules, forexample as described in Antibody Engineering: Methods and Protocols,Second Edition (Humana Press, 2012), at Chapter 40: Production ofBispecific Antibodies: Diabodies and Tandem scFv (Hornig andFärber-Schwarz), or French, How to make bispecific antibodies, MethodsMol. Med. 2000; 40:333-339.

For example, a DNA construct encoding the light and heavy chain variabledomains for the two antigen binding domains (i.e. the light and heavychain variable domains for the antigen binding domain capable of bindingIL-11 or IL-11R, and the light and heavy chain variable domains for theantigen binding domain capable of binding to another target protein),and including sequences encoding a suitable linker or dimerizationdomain between the antigen binding domains can be prepared by molecularcloning techniques. Recombinant bispecific antibody can thereafter beproduced by expression (e.g. in vitro) of the construct in a suitablehost cell (e.g. a mammalian host cell), and expressed recombinantbispecific antibody can then optionally be purified.

Aptamers

Aptamers, also called nucleic acid ligands, are nucleic acid moleculescharacterised by the ability to bind to a target molecule with highspecificity and high affinity. Almost every aptamer identified to dateis a non-naturally occurring molecule.

Aptamers to a given target (e.g. IL-11 or IL-11R) may be identifiedand/or produced by the method of Systematic Evolution of Ligands byEXponential enrichment (SELEX™). Aptamers and SELEX are described inTuerk and Gold (Systematic evolution of ligands by exponentialenrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science.1990 Aug. 3; 249(4968):505-10) and in WO91/19813.

Aptamers may be DNA or RNA molecules and may be single stranded ordouble stranded. The aptamer may comprise chemically modified nucleicacids, for example in which the sugar and/or phosphate and/or base ischemically modified. Such modifications may improve the stability of theaptamer or make the aptamer more resistant to degradation and mayinclude modification at the 2′ position of ribose.

Aptamers may be synthesised by methods which are well known to theskilled person. For example, aptamers may be chemically synthesised,e.g. on a solid support.

Solid phase synthesis may use phosphoramidite chemistry. Briefly, asolid supported nucleotide is detritylated, then coupled with a suitablyactivated nucleoside phosphoramidite to form a phosphite triesterlinkage. Capping may then occur, followed by oxidation of the phosphitetriester with an oxidant, typically iodine. The cycle may then berepeated to assemble the aptamer.

Aptamers can be thought of as the nucleic acid equivalent of monoclonalantibodies and often have K_(d)'s in the nM or pM range, e.g. less thanone of 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500 pM, 100 pM. As withmonoclonal antibodies, they may be useful in virtually any situation inwhich target binding is required, including use in therapeutic anddiagnostic applications, in vitro or in vivo. In vitro diagnosticapplications may include use in detecting the presence or absence of atarget molecule.

Aptamers according to the present invention may be provided in purifiedor isolated form. Aptamers according to the present invention may beformulated as a pharmaceutical composition or medicament.

Suitable aptamers may optionally have a minimum length of one of 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides

Suitable aptamers may optionally have a maximum length of one of 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, or 80 nucleotides

Suitable aptamers may optionally have a length of one of 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides.

Oligonucleotide Repression of IL-11 or IL-11R Expression

Oligonucleotide molecules, particularly RNA, may be employed to regulategene expression. These include antisense oligonucleotides, targeteddegradation of mRNAs by small interfering RNAs (siRNAs), posttranscriptional gene silencing (PTGs), developmentally regulatedsequence-specific translational repression of mRNA by micro-RNAs(miRNAs) and targeted transcriptional gene silencing.

An antisense oligonucleotide is an oligonucleotide, preferably singlestranded, that targets and binds, by complementary sequence binding, toa target oligonucleotide, e.g. mRNA. Where the target oligonucleotide isan mRNA, binding of the antisense to the mRNA blocks translation of themRNA and expression of the gene product. Antisense oligonucleotides maybe designed to bind sense genomic nucleic acid and inhibit transcriptionof a target nucleotide sequence.

In view of the known nucleic acid sequences for IL-11 (e.g. the knownmRNA sequences available from GenBank® under accession no.s: BC012506.1GI:15341754 (human), BC134354.1 GI:126632002 (mouse), AF347935.1GI:13549072 (rat)) and IL-11R (e.g. the known mRNA sequences availablefrom GenBank® under accession no.s: NM_001142784.2 GI:391353394 (human),NM_001163401.1 GI:254281268 (mouse), NM_139116.1 GI:20806172 (rat)),oligonucleotides may be designed to repress or silence the expression ofIL-11 or IL-11R. Such oligonucleotides may have any length, but maypreferably be short, e.g. less than 100 nucleotides, e.g. 10-40nucleotides, or 20-50 nucleotides, and may comprise a nucleotidesequence having complete- or near-complementarity (e.g. 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementarity) toa sequence of nucleotides of corresponding length in the targetoligonucleotide, e.g. the IL-11 or IL-11R mRNA. The complementary regionof the nucleotide sequence may have any length, but is preferably atleast 5, and optionally no more than 50, nucleotides long, e.g. one of6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or 50 nucleotides.

Repression of IL-11 or IL-11R expression will preferably result in adecrease in the quantity of IL-11 or IL-11R expressed by a cell, e.g. bya fibroblast or myofibroblast. For example, in a given cell therepression of IL-11 or IL-11R by administration of a suitable nucleicacid will result in a decrease in the quantity of IL-11 or IL-11Rexpressed by that cell relative to an untreated cell. Repression may bepartial. Preferred degrees of repression are at least 50%, morepreferably one of at least 60%, 70%, 80%, 85% or 90%. A level ofrepression between 90% and 100% is considered a ‘silencing’ ofexpression or function.

A role for the RNAi machinery and small RNAs in targeting ofheterochromatin complexes and epigenetic gene silencing at specificchromosomal loci has been demonstrated. Double-stranded RNA(dsRNA)-dependent post transcriptional silencing, also known as RNAinterference (RNAi), is a phenomenon in which dsRNA complexes can targetspecific genes of homology for silencing in a short period of time. Itacts as a signal to promote degradation of mRNA with sequence identity.A 20-nt siRNA is generally long enough to induce gene-specificsilencing, but short enough to evade host response. The decrease inexpression of targeted gene products can be extensive with 90% silencinginduced by a few molecules of siRNA. RNAi based therapeutics have beenprogressed into Phase I, II and III clinical trials for a number ofindications (Nature 2009 Jan. 22; 457(7228):426-433).

In the art, these RNA sequences are termed “short or small interferingRNAs” (siRNAs) or “microRNAs” (miRNAs) depending on their origin. Bothtypes of sequence may be used to down-regulate gene expression bybinding to complementary RNAs and either triggering mRNA elimination(RNAi) or arresting mRNA translation into protein. siRNA are derived byprocessing of long double stranded RNAs and when found in nature aretypically of exogenous origin. Micro-interfering RNAs (miRNA) areendogenously encoded small non-coding RNAs, derived by processing ofshort hairpins. Both siRNA and miRNA can inhibit the translation ofmRNAs bearing partially complimentary target sequences without RNAcleavage and degrade mRNAs bearing fully complementary sequences.

Accordingly, the present invention provides the use of oligonucleotidesequences for down-regulating the expression of IL-11 or IL-11R.

siRNA ligands are typically double stranded and, in order to optimisethe effectiveness of RNA mediated down-regulation of the function of atarget gene, it is preferred that the length of the siRNA molecule ischosen to ensure correct recognition of the siRNA by the RISC complexthat mediates the recognition by the siRNA of the mRNA target and sothat the siRNA is short enough to reduce a host response.

miRNA ligands are typically single stranded and have regions that arepartially complementary enabling the ligands to form a hairpin. miRNAsare RNA genes which are transcribed from DNA, but are not translatedinto protein. A DNA sequence that codes for a miRNA gene is longer thanthe miRNA. This DNA sequence includes the miRNA sequence and anapproximate reverse complement. When this DNA sequence is transcribedinto a single-stranded RNA molecule, the miRNA sequence and itsreverse-complement base pair to form a partially double stranded RNAsegment. The design of microRNA sequences is discussed in John et al,PLoS Biology, 11(2), 1862-1879, 2004.

Typically, the RNA ligands intended to mimic the effects of siRNA ormiRNA have between 10 and 40 ribonucleotides (or synthetic analoguesthereof), more preferably between 17 and 30 ribonucleotides, morepreferably between 19 and 25 ribonucleotides and most preferably between21 and 23 ribonucleotides. In some embodiments of the inventionemploying double-stranded siRNA, the molecule may have symmetric 3′overhangs, e.g. of one or two (ribo)nucleotides, typically a UU of dTdT3′ overhang. Based on the disclosure provided herein, the skilled personcan readily design suitable siRNA and miRNA sequences, for example usingresources such the Ambion siRNA finder. siRNA and miRNA sequences can besynthetically produced and added exogenously to cause genedownregulation or produced using expression systems (e.g. vectors). In apreferred embodiment the siRNA is synthesized synthetically.

Longer double stranded RNAs may be processed in the cell to producesiRNAs (see for example Myers (2003) Nature Biotechnology 21:324-328).The longer dsRNA molecule may have symmetric 3′ or 5′ overhangs, e.g. ofone or two (ribo)nucleotides, or may have blunt ends. The longer dsRNAmolecules may be 25 nucleotides or longer. Preferably, the longer dsRNAmolecules are between 25 and 30 nucleotides long. More preferably, thelonger dsRNA molecules are between 25 and 27 nucleotides long. Mostpreferably, the longer dsRNA molecules are 27 nucleotides in length.dsRNAs 30 nucleotides or more in length may be expressed using thevector pDECAP (Shinagawa et al., Genes and Dev., 17, 1340-5, 2003).

Another alternative is the expression of a short hairpin RNA molecule(shRNA) in the cell. shRNAs are more stable than synthetic siRNAs. AshRNA consists of short inverted repeats separated by a small loopsequence. One inverted repeat is complimentary to the gene target. Inthe cell the shRNA is processed by DICER into a siRNA which degrades thetarget gene mRNA and suppresses expression. In a preferred embodimentthe shRNA is produced endogenously (within a cell) by transcription froma vector. shRNAs may be produced within a cell by transfecting the cellwith a vector encoding the shRNA sequence under control of a RNApolymerase III promoter such as the human H1 or 7SK promoter or a RNApolymerase II promoter. Alternatively, the shRNA may be synthesisedexogenously (in vitro) by transcription from a vector. The shRNA maythen be introduced directly into the cell. Preferably, the shRNAmolecule comprises a partial sequence of IL-11 or IL-11R. Preferably,the shRNA sequence is between 40 and 100 bases in length, morepreferably between 40 and 70 bases in length. The stem of the hairpin ispreferably between 19 and 30 base pairs in length. The stem may containG-U pairings to stabilise the hairpin structure.

siRNA molecules, longer dsRNA molecules or miRNA molecules may be maderecombinantly by transcription of a nucleic acid sequence, preferablycontained within a vector. Preferably, the siRNA molecule, longer dsRNAmolecule or miRNA molecule comprises a partial sequence of IL-11 orIL-11R.

In one embodiment, the siRNA, longer dsRNA or miRNA is producedendogenously (within a cell) by transcription from a vector. The vectormay be introduced into the cell in any of the ways known in the art.Optionally, expression of the RNA sequence can be regulated using atissue specific (e.g. heart, liver, kidney or eye specific) promoter. Ina further embodiment, the siRNA, longer dsRNA or miRNA is producedexogenously (in vitro) by transcription from a vector.

Suitable vectors may be oligonucleotide vectors configured to expressthe oligonucleotide agent capable of IL-11 or IL-11R repression. Suchvectors may be viral vectors or plasmid vectors. The therapeuticoligonucleotide may be incorporated in the genome of a viral vector andbe operably linked to a regulatory sequence, e.g. promoter, which drivesits expression. The term “operably linked” may include the situationwhere a selected nucleotide sequence and regulatory nucleotide sequenceare covalently linked in such a way as to place the expression of anucleotide sequence under the influence or control of the regulatorysequence. Thus a regulatory sequence is operably linked to a selectednucleotide sequence if the regulatory sequence is capable of effectingtranscription of a nucleotide sequence which forms part or all of theselected nucleotide sequence.

Viral vectors encoding promoter-expressed siRNA sequences are known inthe art and have the benefit of long term expression of the therapeuticoligonucleotide. Examples include lentiviral (Nature 2009 Jan. 22;457(7228):426-433), adenovirus (Shen et al., FEBS Lett 2003 Mar. 27;539(1-3)111-4) and retroviruses (Barton and Medzhitov PNAS Nov. 12, 2002vol. 99, no. 23 14943-14945).

In other embodiments a vector may be configured to assist delivery ofthe therapeutic oligonucleotide to the site at which repression of IL-11or IL-11R expression is required. Such vectors typically involvecomplexing the oligonucleotide with a positively charged vector (e.g.,cationic cell penetrating peptides, cationic polymers and dendrimers,and cationic lipids); conjugating the oligonucleotide with smallmolecules (e.g., cholesterol, bile acids, and lipids), polymers,antibodies, and RNAs; or encapsulating the oligonucleotide innanoparticulate formulations (Wang et al., AAPS J. 2010 December; 12(4):492-503).

In one embodiment, a vector may comprise a nucleic acid sequence in boththe sense and antisense orientation, such that when expressed as RNA thesense and antisense sections will associate to form a double strandedRNA.

Alternatively, siRNA molecules may be synthesized using standard solidor solution phase synthesis techniques which are known in the art.Linkages between nucleotides may be phosphodiester bonds oralternatives, for example, linking groups of the formula P(O)S,(thioate); P(S)S, (dithioate); P(O)NR′2; P(O)R′; P(O)OR6; CO; or CONR′2wherein R is H (or a salt) or alkyl (1-12C) and R6 is alkyl (1-9C) isjoined to adjacent nucleotides through —O— or —S—.

Modified nucleotide bases can be used in addition to the naturallyoccurring bases, and may confer advantageous properties on siRNAmolecules containing them.

For example, modified bases may increase the stability of the siRNAmolecule, thereby reducing the amount required for silencing. Theprovision of modified bases may also provide siRNA molecules which aremore, or less, stable than unmodified siRNA.

The term ‘modified nucleotide base’ encompasses nucleotides with acovalently modified base and/or sugar. For example, modified nucleotidesinclude nucleotides having sugars which are covalently attached to lowmolecular weight organic groups other than a hydroxyl group at the3′position and other than a phosphate group at the 5′position. Thusmodified nucleotides may also include 2′substituted sugars such as2′-O-methyl-; 2′-O-alkyl; 2′-O-allyl; 2′-S-alkyl; 2′-S-allyl;2′-fluoro-; 2′-halo or azido-ribose, carbocyclic sugar analogues,a-anomeric sugars; epimeric sugars such as arabinose, xyloses orlyxoses, pyranose sugars, furanose sugars, and sedoheptulose.

Modified nucleotides are known in the art and include alkylated purinesand pyrimidines, acylated purines and pyrimidines, and otherheterocycles. These classes of pyrimidines and purines are known in theart and include pseudoisocytosine, N4,N4-ethanocytosine,8-hydroxy-N6-methyladenine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil, 5 fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentyl-adenine, 1-methyladenine,1-methylpseudouracil, 1-methylguanine, 2,2-dimethylguanine,2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine,N6-methyladenine, 7-methylguanine, 5-methylaminomethyl uracil, 5-methoxyamino methyl-2-thiouracil, -D-mannosylqueosine,5-methoxycarbonylmethyluracil, 5methoxyuracil, 2methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methyl ester,psueouracil, 2-thiocytosine, 5-methyl-2 thiouracil, 2-thiouracil,4-thiouracil, 5methyluracil, N-uracil-5-oxyacetic acid methylester,uracil 5-oxyacetic acid, queosine, 2-thiocytosine, 5-propyluracil,5-propylcytosine, 5-ethyluracil, 5ethylcytosine, 5-butyluracil,5-pentyluracil, 5-pentylcytosine, and 2,6,diaminopurine,methylpsuedouracil, 1-methylguanine, 1-methylcytosine.

Methods relating to the use of RNAi to silence genes in C. elegans,Drosophila, plants, and mammals are known in the art (Fire A, et al.,1998 Nature 391:806-811; Fire, A. Trends Genet. 15, 358-363 (1999);Sharp, P. A. RNA interference 2001. Genes Dev. 15, 485-490 (2001);Hammond, S. M., et al., Nature Rev. Genet. 2, 110-1119 (2001); Tuschl,T. Chem. Biochem. 2, 239-245 (2001); Hamilton, A. et al., Science 286,950-952 (1999); Hammond, S. M., et al., Nature 404, 293-296 (2000);Zamore, P. D., et al., Cell 101, 25-33 (2000); Bernstein, E., et al.,Nature 409, 363-366 (2001); Elbashir, S. M., et al., Genes Dev. 15,188-200 (2001); WO0129058; WO9932619, and Elbashir S M, et al., 2001Nature 411:494-498).

Accordingly, the invention provides nucleic acid that is capable, whensuitably introduced into or expressed within a mammalian, e.g. human,cell that otherwise expresses IL-11 or IL-11R, of suppressing IL-11 orIL-11R expression by RNAi.

The nucleic acid may have substantial sequence identity to a portion ofIL-11 or IL-11R mRNA, as defined in GenBank accession no. NM_000641.3GI:391353405 (IL-11) or U32324.1 GI:975336 (IL-11R), or thecomplementary sequence to said mRNA.

The nucleic acid may be a double-stranded siRNA. (As the skilled personwill appreciate, and as explained further below, a siRNA molecule mayinclude a short 3′ DNA sequence also.)

Alternatively, the nucleic acid may be a DNA (usually double-strandedDNA) which, when transcribed in a mammalian cell, yields an RNA havingtwo complementary portions joined via a spacer, such that the RNA takesthe form of a hairpin when the complementary portions hybridise witheach other. In a mammalian cell, the hairpin structure may be cleavedfrom the molecule by the enzyme DICER, to yield two distinct, buthybridised, RNA molecules.

In some preferred embodiments, the nucleic acid is generally targeted tothe sequence of one of SEQ ID NOs 2 to 5 (IL-11; FIG. 11) or to one ofSEQ ID NOs 7 to 10 (IL-11R; FIG. 12).

Only single-stranded (i.e. non self-hybridised) regions of an mRNAtranscript are expected to be suitable targets for RNAi. It is thereforeproposed that other sequences very close in the IL-11 or IL-11R mRNAtranscript to the sequence represented by one of SEQ ID NOs 2 to 5 or 7to 10 may also be suitable targets for RNAi. Such target sequences arepreferably 17-23 nucleotides in length and preferably overlap one of SEQID NOs 2 to 5 or 7 to 10 by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18 or all 19 nucleotides (at either end of oneof SEQ ID NOs 2 to 5 or 7 to 10).

Accordingly, the invention provides nucleic acid that is capable, whensuitably introduced into or expressed within a mammalian cell thatotherwise expresses IL-11 or IL-11R, of suppressing IL-11 or IL-11Rexpression by RNAi, wherein the nucleic acid is generally targeted tothe sequence of one of SEQ ID NOs 2 to 5 or 7 to 10.

By “generally targeted” the nucleic acid may target a sequence thatoverlaps with SEQ ID NOs 2 to 5 or 7 to 10. In particular, the nucleicacid may target a sequence in the mRNA of human IL-11 or IL-11R that isslightly longer or shorter than one of SEQ ID NOs 2 to 5 or 7 to 10(preferably from 17-23 nucleotides in length), but is otherwiseidentical to one of SEQ ID NOs 2 to 5 or 7 to 10.

It is expected that perfect identity/complementarity between the nucleicacid of the invention and the target sequence, although preferred, isnot essential. Accordingly, the nucleic acid of the invention mayinclude a single mismatch compared to the mRNA of IL-11 or IL-11R. It isexpected, however, that the presence of even a single mismatch is likelyto lead to reduced efficiency, so the absence of mismatches ispreferred. When present, 3′ overhangs may be excluded from theconsideration of the number of mismatches.

The term “complementarity” is not limited to conventional base pairingbetween nucleic acid consisting of naturally occurring ribo- and/ordeoxyribonucleotides, but also includes base pairing between mRNA andnucleic acids of the invention that include non-natural nucleotides.

In one embodiment, the nucleic acid (herein referred to asdouble-stranded siRNA) includes the double-stranded RNA sequences shownin FIG. 13 (IL-11; SEQ ID NOs 11 to 14).

In another embodiment, the nucleic acid (herein referred to asdouble-stranded siRNA) includes the double-stranded RNA sequences shownin FIG. 14 (IL-11R; SEQ ID NOs 15 to 18).

However, it is also expected that slightly shorter or longer sequencesdirected to the same region of IL-11 or IL-11R mRNA will also beeffective. In particular, it is expected that double-stranded sequencesbetween 17 and 23 bp in length will also be effective.

The strands that form the double-stranded RNA may have short 3′dinucleotide overhangs, which may be DNA or RNA. The use of a 3′ DNAoverhang has no effect on siRNA activity compared to a 3′ RNA overhang,but reduces the cost of chemical synthesis of the nucleic acid strands(Elbashir et al., 2001c). For this reason, DNA dinucleotides may bepreferred.

When present, the dinucleotide overhangs may be symmetrical to eachother, though this is not essential. Indeed, the 3′ overhang of thesense (upper) strand is irrelevant for RNAi activity, as it does notparticipate in mRNA recognition and degradation (Elbashir et al., 2001a,2001b, 2001c).

While RNAi experiments in Drosophila show that antisense 3′ overhangsmay participate in mRNA recognition and targeting (Elbashir et al.2001c), 3′ overhangs do not appear to be necessary for RNAi activity ofsiRNA in mammalian cells. Incorrect annealing of 3′ overhangs istherefore thought to have little effect in mammalian cells (Elbashir etal. 2001c; Czauderna et al. 2003).

Any dinucleotide overhang may therefore be used in the antisense strandof the siRNA. Nevertheless, the dinucleotide is preferably -UU or -UG(or -TT or -TG if the overhang is DNA), more preferably -UU (or -TT).The -UU (or -TT) dinucleotide overhang is most effective and isconsistent with (i.e. capable of forming part of) the RNA polymerase IIIend of transcription signal (the terminator signal is TTTTT).Accordingly, this dinucleotide is most preferred. The dinucleotides AA,CC and GG may also be used, but are less effective and consequently lesspreferred.

Moreover, the 3′ overhangs may be omitted entirely from the siRNA.

The invention also provides single-stranded nucleic acids (hereinreferred to as single-stranded siRNAs) respectively consisting of acomponent strand of one of the aforementioned double-stranded nucleicacids, preferably with the 3′-overhangs, but optionally without. Theinvention also provides kits containing pairs of such single-strandednucleic acids, which are capable of hybridising with each other in vitroto form the aforementioned double-stranded siRNAs, which may then beintroduced into cells.

The invention also provides DNA that, when transcribed in a mammaliancell, yields an RNA (herein also referred to as an shRNA) having twocomplementary portions which are capable of self-hybridising to producea double-stranded motif, e.g. including a sequence selected from thegroup consisting of SEQ ID No.s 11 to 14 or 15 to 18 or a sequence thatdiffers from any one of the aforementioned sequences by a single basepair substitution.

The complementary portions will generally be joined by a spacer, whichhas suitable length and sequence to allow the two complementary portionsto hybridise with each other. The two complementary (i.e. sense andantisense) portions may be joined 5′-3′ in either order. The spacer willtypically be a short sequence, of approximately 4-12 nucleotides,preferably 4-9 nucleotides, more preferably 6-9 nucleotides.

Preferably the 5′ end of the spacer (immediately 3′ of the upstreamcomplementary portion) consists of the nucleotides -UU- or -UG-, againpreferably -UU- (though, again, the use of these particulardinucleotides is not essential). A suitable spacer, recommended for usein the pSuper system of OligoEngine (Seattle, Wash., USA) is UUCAAGAGA.In this and other cases, the ends of the spacer may hybridise with eachother, e.g. elongating the double-stranded motif beyond the exactsequences of SEQ ID NOs 11 to 14 or 15 to 18 by a small number (e.g. 1or 2) of base pairs.

Similarly, the transcribed RNA preferably includes a 3′ overhang fromthe downstream complementary portion. Again, this is preferably -UU or-UG, more preferably -UU.

Such shRNA molecules may then be cleaved in the mammalian cell by theenzyme DICER to yield a double-stranded siRNA as described above, inwhich one or each strand of the hybridised dsRNA includes a 3′ overhang.

Techniques for the synthesis of the nucleic acids of the invention areof course well known in the art.

The skilled person is well able to construct suitable transcriptionvectors for the DNA of the invention using well-known techniques andcommercially available materials. In particular, the DNA will beassociated with control sequences, including a promoter and atranscription termination sequence.

Of particular suitability are the commercially available pSuper andpSuperior systems of OligoEngine (Seattle, Wash., USA). These use apolymerase-III promoter (H1) and a T₅ transcription terminator sequencethat contributes two U residues at the 3′ end of the transcript (which,after DICER processing, provide a 3′ UU overhang of one strand of thesiRNA).

Another suitable system is described in Shin et al. (RNA, 2009 May;15(5): 898-910), which uses another polymerase-III promoter (U6).

The double-stranded siRNAs of the invention may be introduced intomammalian cells in vitro or in vivo using known techniques, as describedbelow, to suppress expression of IL-11 or IL-11R.

Similarly, transcription vectors containing the DNAs of the inventionmay be introduced into tumour cells in vitro or in vivo using knowntechniques, as described below, for transient or stable expression ofRNA, again to suppress expression of IL-11 or IL-11R.

Accordingly, the invention also provides a method of suppressing IL-11or IL-11R expression in a mammalian, e.g. human, cell, the methodcomprising administering to the cell a double-stranded siRNA of theinvention or a transcription vector of the invention.

Similarly, the invention further provides a method of treating fibrosis,the method comprising administering to a subject a double-stranded siRNAof the invention or a transcription vector of the invention.

The invention further provides the double-stranded siRNAs of theinvention and the transcription vectors of the invention, for use in amethod of treatment, preferably a method of treating fibrosis.

The invention further provides the use of the double-stranded siRNAs ofthe invention and the transcription vectors of the invention in thepreparation of a medicament for the treatment of fibrosis.

The invention further provides a composition comprising adouble-stranded siRNA of the invention or a transcription vector of theinvention in admixture with one or more pharmaceutically acceptablecarriers. Suitable carriers include lipophilic carriers or vesicles,which may assist in penetration of the cell membrane.

Materials and methods suitable for the administration of siRNA duplexesand DNA vectors of the invention are well known in the art and improvedmethods are under development, given the potential of RNAi technology.

Generally, many techniques are available for introducing nucleic acidsinto mammalian cells. The choice of technique will depend on whether thenucleic acid is transferred into cultured cells in vitro or in vivo inthe cells of a patient. Techniques suitable for the transfer of nucleicacid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE dextran and calciumphosphate precipitation. In vivo gene transfer techniques includetransfection with viral (typically retroviral) vectors and viral coatprotein-liposome mediated transfection (Dzau et al. (2003) Trends inBiotechnology 11, 205-210).

In particular, suitable techniques for cellular administration of thenucleic acids of the invention both in vitro and in vivo are disclosedin the following articles:

General reviews: Borkhardt, A. 2002. Blocking oncogenes in malignantcells by RNA interference—new hope for a highly specific cancertreatment? Cancer Cell. 2:167-8. Hannon, G. J. 2002. RNA interference.Nature. 418:244-51. McManus, M. T., and P. A. Sharp. 2002. Genesilencing in mammals by small interfering RNAs. Nat Rev Genet. 3:737-47.Scherr, M., M. A. Morgan, and M. Eder. 2003b. Gene silencing mediated bysmall interfering RNAs in mammalian cells. Curr Med Chem. 10:245-56.Shuey, D. J., D. E. McCallus, and T. Giordano. 2002. RNAi:gene-silencing in therapeutic intervention. Drug Discov Today. 7:1040-6.

Systemic delivery using liposomes: Lewis, D. L., J. E. Hagstrom, A. G.Loomis, J. A. Wolff, and H. Herweijer. 2002. Efficient delivery of siRNAfor inhibition of gene expression in postnatal mice. Nat Genet.32:107-8. Paul, C. P., P. D. Good, I. Winer, and D. R. Engelke. 2002.Effective expression of small interfering RNA in human cells. NatBiotechnol. 20:505-8. Song, E., S. K. Lee, J. Wang, N. Ince, N. Ouyang,J. Min, J. Chen, P. Shankar, and J. Lieberman. 2003. RNA interferencetargeting Fas protects mice from fulminant hepatitis. Nat Med. 9:347-51.Sorensen, D. R., M. Leirdal, and M. Sioud. 2003. Gene silencing bysystemic delivery of synthetic siRNAs in adult mice. J Mol Biol.327:761-6.

Virus mediated transfer: Abbas-Terki, T., W. Blanco-Bose, N. Deglon, W.Pralong, and P. Aebischer. 2002. Lentiviral-mediated RNA interference.Hum Gene Ther. 13:2197-201. Barton, G. M., and R. Medzhitov. 2002.Retroviral delivery of small interfering RNA into primary cells. ProcNatl Acad Sci USA. 99:14943-5. Devroe, E., and P. A. Silver. 2002.Retrovirus-delivered siRNA. BMC Biotechnol. 2:15. Lori, F., P. Guallini,L. Galluzzi, and J. Lisziewicz. 2002. Gene therapy approaches to HIVinfection. Am J Pharmacogenomics. 2:245-52. Matta, H., B. Hozayev, R.Tomar, P. Chugh, and P. M. Chaudhary. 2003. Use of lentiviral vectorsfor delivery of small interfering RNA. Cancer Biol Ther. 2:206-10. Qin,X. F., D. S. An, I. S. Chen, and D. Baltimore. 2003. Inhibiting HIV-1infection in human T cells by lentiviral-mediated delivery of smallinterfering RNA against CCR5. Proc Natl Acad Sci USA. 100:183-8. Scherr,M., K. Battmer, A. Ganser, and M. Eder. 2003a. Modulation of geneexpression by lentiviral-mediated delivery of small interfering RNA.Cell Cycle. 2:251-7. Shen, C., A. K. Buck, X. Liu, M. Winkler, and S. N.Reske. 2003. Gene silencing by adenovirus-delivered siRNA. FEBS Lett.539:111-4.

Peptide delivery: Morris, M. C., L. Chaloin, F. Heitz, and G. Divita.2000. Translocating peptides and proteins and their use for genedelivery. Curr Opin Biotechnol. 11:461-6. Simeoni, F., M. C. Morris, F.Heitz, and G. Divita. 2003. Insight into the mechanism of thepeptide-based gene delivery system MPG: implications for delivery ofsiRNA into mammalian cells. Nucleic Acids Res. 31:2717-24. Othertechnologies that may be suitable for delivery of siRNA to the targetcells are based on nanoparticles or nanocapsules such as those describedin U.S. Pat. Nos. 6,649,192B and 5,843,509B.

Formulations

In therapeutic applications, agents capable of inhibiting the action ofIL-11 or agents capable of preventing or reducing the expression ofIL-11 or IL-11R are preferably formulated as a medicament orpharmaceutical together with one or more other pharmaceuticallyacceptable ingredients well known to those skilled in the art,including, but not limited to, pharmaceutically acceptable carriers,adjuvants, excipients, diluents, fillers, buffers, preservatives,anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g.,wetting agents), masking agents, colouring agents, flavouring agents,and sweetening agents.

The term “pharmaceutically acceptable” as used herein pertains tocompounds, ingredients, materials, compositions, dosage forms, etc.,which are, within the scope of sound medical judgment, suitable for usein contact with the tissues of the subject in question (e.g., human)without excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio. Each carrier, adjuvant, excipient, etc. must also be “acceptable”in the sense of being compatible with the other ingredients of theformulation.

Suitable carriers, adjuvants, excipients, etc. can be found in standardpharmaceutical texts, for example, Remington's Pharmaceutical Sciences,18th edition, Mack Publishing Company, Easton, Pa., 1990; and Handbookof Pharmaceutical Excipients, 2nd edition, 1994.

The formulations may be prepared by any methods well known in the art ofpharmacy. Such methods include the step of bringing into association theactive compound with a carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active compound with carriers(e.g., liquid carriers, finely divided solid carrier, etc.), and thenshaping the product, if necessary.

The formulations may be prepared for topical, parenteral, systemic,intravenous, intra-arterial, intramuscular, intrathecal, intraocular,intra-conjunctival, subcutaneous, oral or transdermal routes ofadministration which may include injection. Injectable formulations maycomprise the selected agent in a sterile or isotonic medium.

Administration is preferably in a “therapeutically effective amount”,this being sufficient to show benefit to the individual. The actualamount administered, and rate and time-course of administration, willdepend on the nature and severity of the disease being treated.Prescription of treatment, e.g. decisions on dosage etc, is within theresponsibility of general practitioners and other medical doctors, andtypically takes account of the disorder to be treated, the condition ofthe individual patient, the site of delivery, the method ofadministration and other factors known to practitioners. Examples of thetechniques and protocols mentioned above can be found in Remington'sPharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams &Wilkins.

Fibrosis

As used herein, “fibrosis” refers to the formation of excess fibrousconnective tissue as a result of the excess deposition of extracellularmatrix components, for example collagen. Fibrous connective tissue ischaracterised by having extracellular matrix (ECM) with a high collagencontent. The collagen may be provided in strands or fibers, which may bearranged irregularly or aligned. The ECM of fibrous connective tissuemay also include glycosaminoglycans.

As used herein, “excess fibrous connective tissue” refers to an amountof connective tissue at a given location (e.g. a given tissue or organ,or part of a given tissue or organ) which is greater than the amount ofconnective tissue present at that location in the absence of fibrosis,e.g. under normal, non-pathological conditions. As used herein, “excessdeposition of extracellular matrix components” refers to a level ofdeposition of one or more extracellular matrix components which isgreater than the level of deposition in the absence of fibrosis, e.g.under normal, non-pathological conditions.

The cellular and molecular mechanisms of fibrosis are described in Wynn,J. Pathol. (2008) 214(2): 199-210, and Wynn and Ramalingam, NatureMedicine (2012) 18:1028-1040, which are hereby incorporated by referencein their entirety.

The main cellular effectors of fibrosis are myofibroblasts, whichproduce a collagen-rich extracellular matrix.

In response to tissue injury, damaged cells and leukocytes producepro-fibrotic factors such as TGFβ, IL-13 and PDGF, which activatefibroblasts to αSMA-expressing myofibroblasts, and recruitmyofibroblasts to the site of injury. Myofibroblasts produce a largeamount of extracellular matrix, and are important mediators in aidingcontracture and closure of the wound. However, under conditions ofpersistent infection or during chronic inflammation there can beoveractivation and recruitment of myofibroblasts, and thusover-production of extracellular matrix components, resulting in theformation of excess fibrous connective tissue.

In some embodiments fibrosis may be triggered by pathologicalconditions, e.g. conditions, infections or disease states that lead toproduction of pro-fibrotic factors such as TGFβ1. In some embodiments,fibrosis may be caused by physical injury/stimuli, chemicalinjury/stimuli or environmental injury/stimuli. Physical injury/stimulimay occur during surgery, e.g. iatrogenic causes. Chemicalinjury/stimuli may include drug induced fibrosis, e.g. following chronicadministration of drugs such as bleomycin, cyclophosphamide, amiodarone,procainamide, penicillamine, gold and nitrofurantoin (Daba et al., SaudiMed J 2004 June; 25(6): 700-6). Environmental injury/stimuli may includeexposure to asbestos fibres or silica.

Fibrosis can occur in many tissues of the body. For example, fibrosiscan occur in the liver (e.g. cirrhosis), lungs, kidney, heart, bloodvessels, eye, skin, pancreas, intestine, brain, and bone marrow.Fibrosis may also occur in multiple organs at once.

In embodiments herein, fibrosis may involve an organ of thegastrointestinal system, e.g. of the liver, small intestine, largeintestine, or pancreas. In some embodiments, fibrosis may involve anorgan of the respiratory system, e.g. the lungs. In embodiments,fibrosis may involve an organ of the cardiovascular system, e.g. of theheart or blood vessels. In some embodiments, fibrosis may involve theskin. In some embodiments, fibrosis may involve an organ of the nervoussystem, e.g. the brain. In some embodiments, fibrosis may involve anorgan of the urinary system, e.g. the kidneys. In some embodiments,fibrosis may involve an organ of the musculoskeletal system, e.g. muscletissue.

In some preferred embodiments, the fibrosis is cardiac or myocardialfibrosis, hepatic fibrosis, or renal fibrosis. In some embodimentscardiac or myocardial fibrosis is associated with dysfunction of themusculature or electrical properties of the heart, or thickening of thewalls of valves of the heart. In some embodiments fibrosis is of theatrium and/or ventricles of the heart. Treatment or prevention of atrialor ventricular fibrosis may help reduce risk or onset of atrialfibrillation, ventricular fibrillation, or myocardial infarction.

In some preferred embodiments hepatic fibrosis is associated withchronic liver disease or liver cirrhosis. In some preferred embodimentsrenal fibrosis is associated with chronic kidney disease.

Diseases/conditions characterised by fibrosis in accordance with thepresent invention include but are not limited to: respiratory conditionssuch as pulmonary fibrosis, cystic fibrosis, idiopathic pulmonaryfibrosis, progressive massive fibrosis, scleroderma, obliterativebronchiolitis, Hermansky-Pudlak syndrome, asbestosis, silicosis, chronicpulmonary hypertension, AIDS associated pulmonary hypertension,sarcoidosis, tumor stroma in lung disease, and asthma; chronic liverdisease, primary biliary cirrhosis (PBC), schistosomal liver disease,liver cirrhosis; cardiovascular conditions such as hypertrophiccardiomyopathy, dilated cardiomyopathy (DCM), fibrosis of the atrium,atrial fibrillation, fibrosis of the ventricle, ventricularfibrillation, myocardial fibrosis, Brugada syndrome, myocarditis,endomyocardial fibrosis, myocardial infarction, fibrotic vasculardisease, hypertensive heart disease, arrhythmogenic right ventricularcardiomyopathy (ARMC), tubulointerstitial and glomerular fibrosis,atherosclerosis, varicose veins, cerebral infarcts; neurologicalconditions such as gliosis and Alzheimer's disease; muscular dystrophysuch as Duchenne muscular dystrophy (DMD) or Becker's muscular dystrophy(BMD); gastrointestinal conditions such as Chron's disease, microscopiccolitis and primary sclerosing cholangitis (PSC); skin conditions suchas scleroderma, nephrogenic systemic fibrosis and cutis keloid;arthrofibrosis; Dupuytren's contracture; mediastinal fibrosis;retroperitoneal fibrosis; myelofibrosis; Peyronie's disease; adhesivecapsulitis; kidney disease (e.g., renal fibrosis, nephritic syndrome,Alport's syndrome, HIV associated nephropathy, polycystic kidneydisease, Fabry's disease, diabetic nephropathy, chronicglomerulonephritis, nephritis associated with systemic lupus);progressive systemic sclerosis (PSS); chronic graft versus host disease;diseases of the eye such as Grave's opthalmopathy, epiretinal fibrosis,retinal fibrosis, subretinal fibrosis (e.g. associated with maculardegeneration (e.g. wet age-related macular degeneration (AMD)), diabeticretinopathy, glaucoma, corneal fibrosis, post-surgical fibrosis (e.g. ofthe posterior capsule following cataract surgery, or of the blebfollowing trabeculectomy for glaucoma), conjunctival fibrosis,subconjunctival fibrosis; arthritis; fibrotic pre-neoplastic andfibrotic neoplastic disease; and fibrosis induced by chemical orenvironmental insult (e.g., cancer chemotherapy, pesticides,radiation/cancer radiotherapy).

It will be appreciated that the many of the diseases/conditions listedabove are interrelated. For example, fibrosis of the ventricle may occurpost myocardial infarction, and is associated with DCM, HCM andmyocarditis.

In particular embodiments, the disease/disorder may be one of pulmonaryfibrosis, atrial fibrillation, ventricular fibrillation, hypertrophiccardiomyopathy (HCM), dilated cardiomyopathy (DCM), non-alcoholicsteatohepatitis (NASH), cirrhosis, chronic kidney disease, scleroderma,systemic sclerosis, keloid, cystic fibrosis, Chron's disease,post-surgical fibrosis or retinal fibrosis.

Treatment, prevention or alleviation of fibrosis according to thepresent invention may be of fibrosis that is associated with anupregulation of IL-11, e.g. an upregulation of IL-11 in cells or tissuein which the fibrosis occurs or may occur, or upregulation ofextracellular IL-11 or IL-11R.

Treatment or alleviation of fibrosis may be effective to preventprogression of the fibrosis, e.g. to prevent worsening of the conditionor to slow the rate of development of the fibrosis. In some embodimentstreatment or alleviation may lead to an improvement in the fibrosis,e.g. a reduction in the amount of deposited collagen fibres.

Prevention of fibrosis may refer to prevention of a worsening of thecondition or prevention of the development of fibrosis, e.g. preventingan early stage fibrosis developing to a later, chronic, stage.

Subject

The subject to be treated may be any animal or human. The subject ispreferably mammalian, more preferably human. The subject may be anon-human mammal, but is more preferably human. The subject may be maleor female. The subject may be a patient.

Sample

A sample obtained from a subject may be of any kind. A biological samplemay be taken from any tissue or bodily fluid, e.g. a blood sample,blood-derived sample, serum sample, lymph sample, semen sample, salivasample, synovial fluid sample. A blood-derived sample may be a selectedfraction of a patient's blood, e.g. a selected cell-containing fractionor a plasma or serum fraction. A sample may comprise a tissue sample orbiopsy; or cells isolated from a subject. Samples may be collected byknown techniques, such as biopsy or needle aspirate. Samples may bestored and/or processed for subsequent determination of IL-11 expressionlevels.

Samples may be used to determine the upregulation of IL-11 or IL-11R inthe subject from which the sample was taken.

In some preferred embodiments a sample may be a tissue sample, e.g.biopsy, taken from heart, liver or kidney tissue. In some embodiments asample may be a tissue sample, e.g. biopsy, taken from the eye.

A sample may contain cells, and may preferably contain fibroblastsand/or myofibroblasts. In some embodiments, fibroblasts ormyofibroblasts may be obtained from heart, liver or kidney tissue, e.g.they may be cardiac fibroblasts or cardiac myofibroblasts (e.g. seeColby et al., Circulation Research 2009; 105:1164-1176), hepaticfibroblasts or hepatic myofibroblasts (e.g. see Zeisberg et al., TheJournal of Biological Chemistry, Aug. 10, 2007, 282, 23337-23347;Brenner., Fibrogenesis & Tissue Repair 2012, 5(Suppl 1):517) or renalfibroblasts or renal myofibroblasts (e.g. see Strutz and Zeisberg. JASNNovember 2006 vol. 17 no. 11 2992-2998). In some embodiments,fibroblasts or myofibroblasts may be obtained from eye tissue, e.g. theymay be corneal fibroblasts.

Upregulation of IL-11 or IL-11R Expression

Some aspects and embodiments of the present invention concern detectionof expression of IL-11 or IL-11R, e.g. in a sample obtained from asubject.

In some aspects and embodiments the present invention concerns theupregulation of expression (over-expression) of IL-11 or IL-11R (as aprotein or oligonucleotide encoding the respective IL-11 or IL-11R) anddetection of such upregulation as an indicator of suitability fortreatment with an agent capable of inhibiting the action of IL-11 orwith an agent capable of preventing or reducing the expression of IL-11or IL-11R.

Upregulation of IL-11 or IL-11R expression comprises expression of IL-11or IL-11R at a level that is greater than would normally be expected fora cell or tissue of a given type.

Upregulation may be determined by determining the level of expression ofIL-11 or IL-11R in a cell or tissue. A comparison may be made betweenthe level of IL-11 or IL-11R expression in a cell or tissue sample froma subject and a reference level of IL-11 or IL-11R, e.g. a value orrange of values representing a normal level of expression of IL-11 orIL-11R for the same or corresponding cell or tissue type. In someembodiments reference levels may be determined by detecting IL-11 orIL-11R expression in a control sample, e.g. in corresponding cells ortissue from a healthy subject or from healthy tissue of the samesubject. In some embodiments reference levels may be obtained from astandard curve or data set.

Levels of expression may be quantitated for absolute comparison, orrelative comparisons may be made.

In some embodiments upregulation of IL-11 or IL-11R may be considered tobe present when the level of expression in the test sample is at least1.1 times that of a reference level. More preferably, the level ofexpression may be selected from one of at least 1.2, at least 1.3, atleast 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, atleast 1.9, at least 2.0, at least 2.1, at least 2.2, at least 2.3, atleast 2.4 at least 2.5, at least 2.6, at least 2.7, at least 2.8, atleast 2.9, at least 3.0, at least 3.5, at least 4.0, at least 5.0, atleast 6.0, at least 7.0, at least 8.0, at least 9.0, or at least 10.0times that of the reference level.

IL-11 or IL-11R expression levels may be determined by one of a numberof known in vitro assay techniques, such as PCR based assays, in situhybridisation assays, flow cytometry assays, immunological orimmunohistochemical assays.

By way of example suitable techniques involve a method of detecting thelevel of IL-11 or IL-11R in a sample by contacting the sample with anagent capable of binding IL-11 or IL-11R and detecting the formation ofa complex of the agent and IL-11 or IL-11R. The agent may be anysuitable binding molecule, e.g. an antibody, polypeptide, peptide,oligonucleotide, aptamer or small molecule, and may optionally belabelled to permit detection, e.g. visualisation, of the complexesformed. Suitable labels and means for their detection are well known tothose in the art and include fluorescent labels (e.g. fluorescein,rhodamine, eosine and NDB, green fluorescent protein (GFP), chelates ofrare earths such as europium (Eu), terbium (Tb) and samarium (Sm),tetramethyl rhodamine, Texas Red, 4-methyl umbelliferone,7-amino-4-methyl coumarin, Cy3, Cy5), isotope markers, radioisotopes(e.g. ³²P, ³³P, ³⁵S), chemiluminescence labels (e.g. acridinium ester,luminol, isoluminol), enzymes (e.g. peroxidase, alkaline phosphatase,glucose oxidase, beta-galactosidase, luciferase), antibodies, ligandsand receptors. Detection techniques are well known to those of skill inthe art and can be selected to correspond with the labelling agent.Suitable techniques include PCR amplification of oligonucleotide tags,mass spectrometry, detection of fluorescence or colour, e.g. uponenzymatic conversion of a substrate by a reporter protein, or detectionof radioactivity.

Assays may be configured to quantify the amount of IL-11 or IL-11R in asample. Quantified amounts of IL-11 or IL-11R from a test sample may becompared with reference values, and the comparison used to determinewhether the test sample contains an amount of IL-11 or IL-11R that ishigher or lower than that of the reference value to a selected degree ofstatistical significance.

Quantification of detected IL-11 or IL-11R may be used to determine up-or down-regulation or amplification of genes encoding IL-11 or IL-11R.In cases where the test sample contains fibrotic cells, suchup-regulation, down-regulation or amplification may be compared to areference value to determine whether any statistically significantdifference is present.

Subject Selection

A subject may be selected for treatment based on a determination thatthe subject has an upregulated level of IL-11 or IL-11R expression.IL-11 or IL-11R may therefore act as a marker of a fibrosis that issuitable for treatment with an agent capable of inhibiting the action ofIL-11 or with an agent capable of preventing or reducing the expressionof IL-11 or IL-11R.

Upregulation may be in a given tissue or in selected cells from a giventissue. A preferred tissue may be one of heart, liver or kidney. Apreferred tissue may be eye. A preferred cell type may be fibroblasts ormyofibroblasts. Upregulation may also be determined in a circulatingfluid, e.g. blood, or in a blood derived sample. Upregulation of may beof extracellular IL-11 or IL-11R.

Determination of IL-11 or IL-11R levels may be performed by assay,preferably in vitro, on a sample obtained from a subject, as describedherein.

Following selection, a subject may be provided with treatment forfibrosis by administration of an agent capable of inhibiting the actionof IL-11 or an agent capable of preventing or reducing the expression ofIL-11 or IL-11R.

In some embodiments a subject may have been diagnosed with fibrosis, besuspected of having fibrosis or be considered at risk of developingfibrosis and it is of interest whether the subject will benefit fromtreatment with an agent capable of inhibiting the action of IL-11 orwith an agent capable of preventing or reducing the expression of IL-11or IL-11R. In such embodiments, the suitability of the subject for suchtreatment may be determined by determining whether IL-11 or IL-11Rexpression is upregulated in the subject. In some embodiments, IL-11 orIL-11R expression is locally or systemically upregulated in the subject.

Diagnosis and Prognosis

The detection of upregulation of IL-11 or IL-11R expression may also beused in a method of diagnosing fibrosis or the risk of developingfibrosis in a subject, and in methods of prognosing or predicting asubject's response to treatment with an agent capable of inhibiting theaction of IL-11 or an agent capable of preventing or reducing theexpression of IL-11 or IL-11R.

In some embodiments a subject may be suspected of having fibrosis, e.g.based on the presence of other symptoms indicative of fibrosis in thesubject's body or in selected cells/tissues of the subject's body, or beconsidered at risk of developing fibrosis, e.g. because of geneticpredisposition or exposure to environmental conditions, such as asbestosfibres.

Determination of upregulation of IL-11 or IL-11R may confirm a diagnosisor suspected diagnosis of fibrosis or may confirm that the subject is atrisk of developing fibrosis. The determination may also diagnose thecondition or predisposition as one suitable for treatment with an agentcapable of inhibiting the action of IL-11 or an agent capable ofpreventing or reducing the expression of IL-11 or IL-11R.

As such, a method of providing a prognosis for a subject having, orsuspected of having fibrosis may be provided, the method comprisingdetermining whether IL-11 or IL-11R is upregulated in a sample obtainedfrom the subject and, based on the determination, providing a prognosisfor treatment of the subject with an agent capable of inhibiting theaction of IL-11 or an agent capable of preventing or reducing theexpression of IL-11 or IL-11R.

In some aspects methods of diagnosis or methods of prognosing orpredicting a subject's response to treatment with an agent capable ofinhibiting the action of IL-11 or an agent capable of preventing orreducing the expression of IL-11 or IL-11R may not require determinationof IL-11 or IL-11R levels, but may be based on determining geneticfactors in the subject that are predictive of upregulation of IL-11 orIL-11R expression, or upregulation of IL-11 or IL-11R activity. Suchgenetic factors may include the determination of genetic mutations,single nucleotide polymorphisms (SNPs) or gene amplification in IL-11and/or IL-11R that are correlated with and/or predictive of upregulationof IL-11 or IL-11R expression or activity or IL-11 mediated signalingactivity. The use of genetic factors to predict predisposition to adisease state or response to treatment is known in the art, e.g. seePeter Stärkel Gut 2008; 57:440-442; Wright et al., Mol. Cell. Biol.March 2010 vol. 30 no. 6 1411-1420.

Genetic factors may be assayed by methods known to those of ordinaryskill in the art, including PCR based assays, e.g. quantitative PCR,competitive PCR. By determining the presence of genetic factors, e.g. ina sample obtained from a subject, a diagnosis of fibrosis may beconfirmed, and/or a subject may be classified as being at risk ofdeveloping fibrosis, and/or a subject may be identified as beingsuitable for treatment with an agent capable of inhibiting the action ofIL-11 or an agent capable of preventing or reducing the expression ofIL-11 or IL-11R.

Some methods may comprise determination of the presence of one or moreSNPs linked to secretion of IL-11 or susceptibility to development offibrosis. SNPs are usually bi-allelic and therefore can be readilydetermined using one of a number of conventional assays known to thoseof skill in the art (e.g. see Anthony J. Brookes. The essence of SNPs.Gene Volume 234, Issue 2, 8 Jul. 1999, 177-186; Fan et al., HighlyParallel SNP Genotyping. Cold Spring Harb Symp Quant Biol 2003. 68:69-78; Matsuzaki et al., Parallel Genotyping of Over 10,000 SNPs using aone-primer assay on a high-density oligonucleotide array. Genome Res.2004. 14: 414-425).

The methods may comprise determining which SNP allele is present in asample obtained from a subject. In some embodiments determining thepresence of the minor allele may be associated with increased IL-11secretion or susceptibility to development of fibrosis.

Accordingly, in one aspect of the present invention a method forscreening a subject is provided, the method comprising:

-   -   obtaining a nucleic acid sample from the subject;    -   determining which allele is present in the sample at the        polymorphic nucleotide position of one or more of the SNPs        listed in FIG. 33, and/or FIG. 34 and/or FIG. 35 or an SNP in        linkage disequilibrium with one of the listed SNPs with an        r²≥0.8.

The determining step may comprise determining whether the minor alleleis present in the sample at the selected polymorphic nucleotideposition. It may comprise determining whether 0, 1 or 2 minor allelesare present.

The screening method may be, or form part of, a method for determiningsusceptibility of the subject to development of fibrosis, or a method ofdiagnosis or prognosis as described herein.

The method may further comprise the step of identifying the subject ashaving susceptibility to, or an increased risk of, developing fibrosis,e.g. if the subject is determined to have a minor allele at thepolymorphic nucleotide position. The method may further comprise thestep of selecting the subject for treatment with an agent capable ofinhibiting the action of Interleukin 11 (IL-11) and/or administering anagent capable of inhibiting the action of Interleukin 11 (IL-11) to thesubject in order to provide a treatment for fibrosis in the subject orto prevent development or progression of fibrosis in the subject.

SNPs that may be determined include one or more of the SNPs listed inFIG. 33, FIG. 34, or FIG. 35. In some embodiments the method maycomprise determining one or more of the SNPs listed in FIG. 33. In someembodiments the method may comprise determining one or more of the SNPslisted in FIG. 34. In some embodiments the method may comprisedetermining one or more of the SNPs listed in FIG. 35. SNPs may beselected for determination as having a low P value or FDR (falsediscovery rate).

In some embodiments SNPs are selected as being good predictors ofresponse to anti-IL-11 treatment based on regulation of VSTstim in trans(FIG. 33). In some embodiments a method may comprise determining whichallele is present for one or more of the following SNPs: rs10831850,rs4756936, rs6485827, rs7120273, and rs895468. In some embodiments SNPsare selected as being good predictors of response to anti-IL-11treatment based on regulation VSTstim−VSTunstim in cis (FIG. 34).

In some embodiments SNPs are selected as being good predictors ofresponse to anti-IL-11 treatment based on regulation VSTstim−VSTunstimin trans (FIG. 35). In some embodiments a method may comprisedetermining which allele is present for one or more of the followingSNPs: rs7120273, rs10831850, rs4756936, rs6485827 (FIG. 35).

SNPs: rs7120273, rs10831850, rs4756936, rs6485827 are in high linkagedisequilibrium (LD) with one another on chromosome 11 (in a so-called LDblock), and are therefore very commonly co-inherited.

The square of the correlation of gene frequencies (r²) reflects thedegree of linkage disequilibrium (LD) between two SNPs. As a result ofLD between SNPs in local and therefore co-inherited regions of thegenome, the genotype of a given SNP can be inferred by determining thegenotype of a tagging/proxy SNP. The threshold of LD used in the art toidentify pairwise tagging/proxy SNPs is an r² value of 0.8 (Wang et al.2005, Nat. Rev. Genet. 6(2): 109-18; Barrett et al. 2006, Nat Genet., 38(6): 659-662). The genotype of a given SNP can therefore be inferred bydetermining the genotype of a tagging/proxy SNP in linkagedisequilibrium with an r² value≥0.8.

The nucleotide sequence of SNPs is indicated using the “rs” number. Thefull sequence is available from the National Center for biotechnologyInformation (NCBI) database of single nucleotide polymorphisms (dbSNP)accessible at: https://www.ncbi.nlm.nih.gov/snp.

Methods of diagnosis or prognosis may be performed in vitro on a sampleobtained from a subject, or following processing of a sample obtainedfrom a subject. Once the sample is collected, the patient is notrequired to be present for the in vitro method of diagnosis or prognosisto be performed and therefore the method may be one which is notpractised on the human or animal body.

Other diagnostic or prognostic tests may be used in conjunction withthose described here to enhance the accuracy of the diagnosis orprognosis or to confirm a result obtained by using the tests describedhere.

Methods according to the present invention may be performed, or productsmay be present, in vitro, ex vivo, or in vivo. The term “in vitro” isintended to encompass experiments with materials, biological substances,cells and/or tissues in laboratory conditions or in culture whereas theterm “in vivo” is intended to encompass experiments and procedures withintact multi-cellular organisms. “Ex vivo” refers to something presentor taking place outside an organism, e.g. outside the human or animalbody, which may be on tissue (e.g. whole organs) or cells taken from theorganism.

The invention includes the combination of the aspects and preferredfeatures described except where such a combination is clearlyimpermissible or expressly avoided.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Aspects and embodiments of the present invention will now beillustrated, by way of example, with reference to the accompanyingfigures. Further aspects and embodiments will be apparent to thoseskilled in the art. All documents mentioned in this text areincorporated herein by reference.

Throughout this specification, including the claims which follow, unlessthe context requires otherwise, the word “comprise,” and variations suchas “comprises” and “comprising,” will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Ranges may be expressedherein as from “about” one particular value, and/or to “about” anotherparticular value. When such a range is expressed, another embodimentincludes from the one particular value and/or to the other particularvalue. Similarly, when values are expressed as approximations, by theuse of the antecedent “about,” it will be understood that the particularvalue forms another embodiment.

EXAMPLES Example 1

The fibrotic response is characterized by widespread molecular changesin activated resident fibroblasts. To establish the role of IL-11 as akey marker of this transition we assessed and ranked global RNAexpression differences in atrial fibroblasts derived from 80 individualsbefore and 24 hours after Transforming growth factor beta-1 (TGFβ1)activation. We cultured primary fibroblasts derived from the atrium of80 individuals who were undergoing cardiac surgery for coronary arterydisease. Fibroblasts were studied ex vivo at baseline and followingstimulation with TGFβ1 (a powerful pro-fibrotic stimulus) usinggenome-wide expression profiling (RNA-Seq) combined with phenotypicassays and genotyping.

IL-11 expression was significantly induced in response to TGFβ1treatment with RNA levels increasing as much 30× (>8× on average). IL-11expression was higher than expression of all other individual genes(FIGS. 1a,b ), meaning that of the ˜11,500 genes expressed infibroblasts IL-11 is the most markedly upregulated. This upregulationIL-11 was confirmed with RT-qPCR as well as ELISA experiments (FIGS.1c,d ), indicating increased production and release of IL-11 protein inactivated fibroblasts is the main drivers of fibrosis.

To assess whether IL-11 acts as an autocrine signaling factor thatdrives fibrosis, we incubated non-stimulated atrial fibroblasts withrecombinant IL-11 and monitored cell proliferation, myofibroblastgeneration as well as collagen and periostin expression at the proteinlevel. We observed an increase in collagen production, cellproliferation and periostin expression at levels similar to thoseinduced by the TGFβ1 signaling pathway. IL-11 activated fibroblasts alsodifferentiated into α-SMA+ myofibroblasts (FIG. 2).

In addition to its pro-fibrotic function, IL-11 was also found to play acritical role in the TGFβ1 induced fibrotic response itself. Inhibitionof IL-11 with a neutralising anti-human IL-11 monoclonal antibody(Monoclonal Mouse IgG_(2A); Clone #22626; Catalog No. MAB218; R&DSystems, MN, USA) reduced the activation of fibroblasts through TGFβ1.Cells incubated with TGFβ1 did not generate more extracellular matrixproteins when the IL-11 antibody was present (FIG. 3).

We showed that IL-11 neutralizing antibodies prevent TGFβ1-inducedfibroblast activation.

Example 2

Inflammation and tissue damage stimulates a dynamic process thatinvolves the recruitment, proliferation and activation of fibroblasts togenerate extracellular matrix and initiate wound healing and scarring.This fibrotic response is characterized by widespread molecular changesin activated resident fibroblasts that can be induced by TGFβ1, amultifunctional cytokine that is released by local and infiltratingcells.

To identify key markers of this transition we assessed and ranked globalRNA expression differences via transcriptome sequencing in atrialfibroblasts derived from 80 individuals before and 24 hours after TGFβ1treatment. As discussed in Example 1, IL-11 expression was significantlyupregulated in activated fibroblasts and we showed for the first timethat the IL-11 transcriptional response is higher than thetranscriptional response of all other individual genes regulated infibrosis (FIG. 4a ). Comparison of the IL-11 expression level in ourmodel system to various human tissues indicated that high IL-11 levelswere also very specific for the fibrotic response (FIG. 4b ), making itan ideal marker to assess the extent of fibrosis in the human body.

To further assess whether IL-11 acts as an autocrine signaling factorthat drives fibrosis, we confirmed that an upregulation of IL-11 RNA(FIG. 5a ) lead to an increase in IL-11 secretion (FIG. 5b ) from atrialfibroblasts. Incubation of fibroblasts with IL-11 did not increase IL-11RNA expression (FIG. 5c ), but lead to an increase in IL-11 secretionfrom the cells (FIG. 5d ). This shows that IL-11 is having an autocrineeffect on fibroblasts that regulates the production of IL-11 protein atthe translational level.

We then incubated atrial fibroblasts with TGFβ1, recombinant IL-11 orTGFβ1 and a neutralising anti- human IL-11 monoclonal antibody(Monoclonal Mouse IgG_(2A); Clone #22626; Catalog No. MAB218; R&DSystems, MN, USA) and monitored cell proliferation, myofibroblastgeneration as well as periostin expression at the protein level. Weobserved an increase in activated fibroblasts (αSMA-positive cells),periostin production and cell proliferation at a similar level for bothTGFβ1 and IL-11 stimulated fibroblasts. In addition to its pro-fibroticfunction, IL-11 was also found to play a critical role in the TGFβ1fibrosis itself. The pro-fibrotic effect of TGFβ1 was inhibited when weneutralized IL-11 with the antibody (FIGS. 6a-c ). The same pattern wasobserved when we monitored the secretion of fibrosis markers such asIL6, MMP2 and TIMP1 (FIGS. 6d-f ).

We then monitored the deposition of collagen, the pathognomonic hallmarkof the fibrotic response, using a number of assays across severalregulatory levels of gene expression. TGFβ1 was found to increaseintracellular collagen (FIG. 7a ), secreted collagen (FIG. 7b ) as wellas collagen RNA levels (FIG. 7c ) as expected. The response to IL-11 wasonly observed at the protein level (FIG. 7a,b ) and not on the RNA level(FIG. 7c ). Stimulation with TGFβ1 in parallel to inhibiting IL-11 ledto an increase in collagen RNA but this TGFβ1-driven effect was notforwarded to the protein level.

To establish further the central role of IL-11 in fibrosis downstream ofmultiple pro-fibrotic stimuli, we assessed IL-11 expression acrossfibroblast populations derived from four different tissues in responseto TGFβ1 (FIG. 8a ), ET-1, (FIG. 8b ) and PDGF (FIG. 8c ). We alsoadministered recombinant IL-11 systemically to C57BL/6 mice andmonitored collagen and αSMA expression. Collagen production wasincreased across kidney, heart and liver (FIG. 8d ) and we also detectedmore activated fibroblasts in the heart and kidney, indicated by higherαSMA protein levels (FIG. 8e ).

Our findings demonstrate a novel and central role for IL-11 in fibrosisand, most importantly, show that IL-11 is downstream of the keypro-fibrotic stimuli across several tissues. These results show thatIL-11 is required for TGFβ1 to proceed from transcriptional regulationto protein translation. Inhibition of IL-11 stalls the pro-fibroticeffect of TGFβ1 on the transcriptome (FIG. 9).

Example 3 Anti-IL-11 Antibodies Inhibit Pro-Fibrotic Stimuli

In experiments similar to those described in respect of FIG. 3c , atrialfibroblasts were exposed to other pro-fibrotic stimuli in the form ofangiotensin II (ANG 2), platelet-derived growth factor (PDGF) andendothelin 1 (ET-1), and collagen production was measured.

In addition to induction of IL-11 mRNA expression, each of ANG2, PDGFand ET-1 induced IL-11 protein expression. Inhibition of IL-11 with aneutralising anti-human IL-11 monoclonal antibody (Monoclonal MouseIgG_(2A); Clone #22626; Catalog No. MAB218; R&D Systems, MN, USA)blocked the pro-fibrotic effect of each of these pro-fibrotic stimuli(FIG. 10) indicating IL-11 to be the central effector of the majorpro-fibrotic stimuli (TGFβ1, ANG2, PDGF and ET-1).

Example 4 IL-11R Knockdown

HEK cells were transfected (24 h) with non-targeting (NT) siRNA or oneof four different siRNAs against the IL11RA1 receptor (siRNAs 5-8; FIG.14; SEQ ID NOs 15 to 18). RNA was extracted and assayed for IL11RA1 mRNAexpression by qPCR. Data are shown in FIG. 15 as mRNA expression levelsrelative to the control (NT).

Example 5 A Role for IL-11 in Fibrosis

5.1 IL-11 is Upregulated in Fibrosis

To understand the molecular processes underlying the transition offibroblasts to activated myofibroblasts, atrial tissue was obtained frommore than 200 patients that underwent cardiac bypass surgery at theNational Heart Centre Singapore. Cells were cultured in vitro at lowpassage (passage<4), and either not stimulated or stimulated with TGFβ1for 24 h. We subsequently performed high-throughput RNA sequencing(RNA-seq) analysis of unstimulated fibroblasts and cells stimulated withthe prototypic pro-fibrotic stimulus TGFβ1 across 160 individuals;average read depth was ˜70M reads per sample (paired-end 100 bp; FIG.16).

To ensure the purity of the atrial fibroblast cell cultures, we analysedexpression of endothelial cell, cardiomyocyte and fibroblast cell typemarker genes from the atrium (Hsu et al., 2012 Circulation CardiovascGenetics 5, 327-335) in the RNA-seq dataset.

The results are shown in FIGS. 17A to 17E, and confirm the purity of theatrial fibroblast cultures.

Gene expression was assessed by RNA-seq of the tissue of origin (humanatrial tissues samples, n=8) and primary, unstimulated fibroblastcultures. No/very low expression of the endothelial cell marker PECAM1(FIG. 17A), and the cardiomyocyte markers MYH6 (FIG. 17B) and TNNT2(FIG. 17C) was detected in the fibroblast cell culture samples. Markersfor fibroblasts COL1A2 (FIG. 17D) and ACTA2 (FIG. 17E) were highlyexpressed compared to the tissue of origin.

Next, the RNA-seq data was analysed to identify genes whose expressionwas increased or decreased upon stimulation with TGFβ1, and thisinformation was integrated with the large RNA-seq dataset across 35+human tissues provided by the GTEx project (The GTEx Consortium, 2015Science 348, 648-660). This enabled the identification of geneexpression signatures that were specific to the fibroblast-myofibroblasttransition.

The results are shown in FIGS. 18A to 18E. Across the 10000+ genesexpressed in the fibroblasts, IL-11 was the most strongly upregulatedgene in response to stimulation with TGFβ1, and on average across the160 individuals was upregulated more than 10-fold (FIG. 18A).

Upregulation of IL-11 expression was confirmed by ELISA analysis of thecell culture supernatant of TGFβ1 stimulated fibroblasts (FIG. 18C). Ascompared to the level of expression level of IL-11 in other tissues ofhealthy individuals, this response was observed to be highly specific toactivated fibroblasts (FIG. 18D). Various fold changes of IL-11 RNAexpression were also confirmed by qPCR analysis (FIG. 18E).

Next, fibroblasts were cultured in vitro and stimulated with severalother known pro-fibrotic factors: ET-1, ANGII, PDGF, OSM and IL-13, andalso with human recombinant IL-11. For analysing upregulation of IL-11produced in response to stimulation with IL-11, it was confirmed thatthe ELISA was only able to detect native IL-11 secreted from cells anddoes not detect recombinant IL-11 used for the stimulations (FIG. 19B).

The results are shown in FIG. 19A. Each factor was found tosignificantly induce IL-11 secretion from fibroblasts. IL-11 is shown toact in an autocrine loop in fibroblasts, which can result in anupregulation of IL-11 protein as much as 100-fold after 72 hours (FIG.19D).

Interestingly, this autocrine loop for IL-11 is similar to the autocrineproduction of IL-6. IL-6 is from the same cytokine family and alsosignals via the gp130 receptor (Garbers and Scheller, 2013 Biol Chem394, 1145-1161), which is proposed to ensure the continued survival andgrowth of lung and breast cancer cells (Grivennikov and Karin, 2008Cancer Cell 13, 7-9).

No increase in IL-11 RNA level was detected in response to stimulationwith IL-11 (FIG. 19D). Unlike TGFβ1, which increases IL-11 expression atboth the RNA and protein level, therefore IL-11 seems to upregulateIL-11 expression only at the post-transcriptional level.

5.2 IL-11 has a Profibrotic Role in Fibrosis of Heart Tissue

To explore whether the autocrine production of IL-11 is pro- oranti-fibrotic, fibroblasts were cultured in vitro with recombinantIL-11, and the fraction of myofibroblasts (αSMA-positive cells) andextracellular matrix production was analysed.

The expression of αSMA, collagen and periostin was monitored with theOperetta High-Content Imaging System in an automated, high-throughputfashion. In parallel, secretion of fibrosis marker proteins such asMMP2, TIMP1 and IL-6 was analysed by ELISA assays, and the levels ofcollagen were confirmed by calorimetric Sirius Red analysis of the cellculture supernatant.

Briefly, atrial fibroblasts derived from 3 individuals were incubated in2 wells each for 24 h without stimulation, with TGFβ1 (5 ng/ml), or withIL-11 (5 ng/ml). Following incubation, cells were stained to analyseα-SMA content to estimate the fraction of myofibroblasts, and forcollagen and periostin to estimate ECM production. Fluorescence wasmeasured in 7 fields per well. The supernatant of 2 wells per individualwas also assessed for collagen content by Sirius Red staining. Thesignal was normalized to the control group without stimulation.Secretion of the fibrosis markers IL-6, TIMP1 and MMP2 was analysed viaELISA.

The results are shown in FIGS. 20A to 20F. TGFβ1 activated fibroblastsand increased ECM production (FIG. 20A). Unexpectedly, and in contrastwith the anti-fibrotic role described for IL-11 in heart tissue in thescientific literature, recombinant IL-11 caused an increase in thefraction of myofibroblasts in fibroblast cultures, and also promoted theproduction of extracellular matrix proteins collagen and periostin tothe same extent as TGFβ1 (FIG. 20A). Both of IL-11 and TGFβ1 cytokinesalso significantly increased the secretion of pro-fibrotic markers IL-6,TIMP1 and MMP2 (FIGS. 20B to 20E), and to a similar level.

The inventors hypothesized that the contradiction between the presentfinding that IL-11 is profibrotic in heart tissue and the antifibroticrole described in the literature might be related to the use of humanIL-11 in rodents in those previous studies (Obana et al., 2010, 2012;Stangou et al., 2011; Trepicchio and Dorner, 1998).

To investigate this hypothesis, serial dilutions of both human and mouseIL-11 were performed, and the activation of human atrial fibroblasts wasmonitored (FIG. 20F). No activation of fibroblasts was observed at lowconcentrations of human IL-11 on mouse cells, suggesting that previousinsights into IL-11 function may in part be due to IL-11-non-specificobservations.

5.3 IL-11 has a Profibrotic Role in Fibrosis of a Variety of Tissues

To test whether the profibrotic action of IL-11 was specific to atrialfibroblasts, human fibroblasts derived from several different tissues(heart, lung, skin, kidney and liver) were cultured in vitro, stimulatedwith human IL-11, and fibroblast activation and ECM production wasanalysed as described above. Increased fibroblast activation andproduction of ECM was observed as compared to non-stimulated cultures infibroblasts derived from each of the tissues analysed.

5.3.1 Liver Fibrosis

To test whether IL-11 signaling is important in liver fibrosis, humanprimary liver fibroblasts (Cell Biologics, Cat#: H-6019) were culturedat low passage in wells of 96-well plates and either not stimulated,stimulated with TGFβ1 (5 ng/ml, 24 h), IL-11 (5 ng/ml, 24 h) orincubated with both TGFβ1 (5 ng/ml) and a neutralising IL-11 antibody (2μg/ml), or TGFβ1 (5 ng/ml) and an Isotype control antibody. Fibroblastactivation (αSMA positive cells), cell proliferation (EdU positivecells) and ECM production (periostin and collagen) was analysed usingthe Operetta platform.

The results of the experiments with primary human liver fibroblasts areshown in FIGS. 38A to 38D. IL-11 was found to activate liverfibroblasts, and IL-11 signaling was found to be necessary for theprofibrotic action of TGFβ1 in liver fibroblasts. Both activation andproliferation of fibroblasts was inhibited by neutralising anti-IL-11antibody.

5.3.2 Skin Fibrosis

To test whether IL-11 signaling is important in skin fibrosis, primarymouse skin fibroblasts were cultured at low passage in wells of 96-wellplates and either not stimulated, stimulated with TGFβ1 (5 ng/ml, 24 h)or incubated for 24 h with both TGFβ1 (5 ng/ml) and a neutralising IL-11antibody (2 μg/ml). Fibroblast activation (αSMA positive cells) was thenanalysed using the Operetta platform.

The results are shown in FIG. 39. TGFβ1-mediated activation of skinfibroblasts was inhibited by neutralising anti-IL-11 antibody.

5.3.3 Fibrosis in Multiple Organs

Next, mouse recombinant IL-11 was injected (100 μg/kg, 3 days/week, 28days) into mice to test whether IL-11 can drive global tissue fibrosisin vivo.

The results are shown in FIG. 21. Compared to injection of AngII (acytokine that causes an elevation in blood pressure and hypertrophy ofthe heart), IL-11 also increased the heart weight but also kidney, lungand liver weight indexed to body weight (FIG. 21B). Assessing collagencontent in these issues by hydroxyproline assay revealed an upregulationof collagen production in these tissues, indicating fibrosis as thelikely cause for the increase in organ weight (FIG. 6C). Expression offibrosis marker genes ACTA2 (=αSMA), Col1a1, Col3a1, Fn1, Mmp2 and Timp1was also detected by qPCR analysis of RNA isolated from heart, kidney,lung and liver tissues of these animals.

Example 6 Therapeutic Potential of IL-11/IL-11R Antagonism

6.1 Inhibition of the Fibrotic Response Using Neutralising Antagonistsof IL-11/IL-11R

Next it was investigated whether the autocrine loop of IL-11 secretionwas required for the pro-fibrotic effect of TGFβ1 on fibroblasts.

IL-11 was inhibited using a commercially available neutralizing antibody(Monoclonal Mouse IgG2A; Clone #22626; Catalog No. MAB218; R&D Systems,MN, USA). Fibroblasts were treated with TGFβ1 in the presence or absenceof the antibody, and fibroblast activation, the proportion ofproliferating cells and ECM production and markers of the fibroticresponse were measured.

Briefly, atrial fibroblasts derived from 3 individuals were incubatedfor 24 h with TGFβ1 (5 ng/ml) or TGFβ1 in the presence of neutralisinganti-IL-11 antibody or isotype control antibody. Following incubation,cells were stained for αSMA to determine the fraction of myofibroblasts,the proportion of proliferating cells was determined by analysing thecells for EdU incorporation, and periostin was measured to determine ECMproduction. Fluorescence was measured with the Operetta platform for 14fields across 2 wells for each individual. Secretion of the fibrosismarkers IL-6, TIMP1 and MMP2 was also analysed by ELISA. Fluorescencewas normalized to the control group without stimulation.

The results are shown in FIGS. 22A to 22F. IL-11 inhibition was found toameliorate TGFβ1-induced fibrosis, and it was shown that IL-11 isessential for the pro-fibrotic effect of TGFβ1. Inhibition of IL-11 wasfound to ‘rescue’ the TGFβ1 phenotype at the protein level.

Collagen production was also analysed. Cardiac fibroblasts derived from3 individuals were incubated for 24 h with TGFβ1 (5 ng/ml) or TGFβ1 anda neutralizing IL-11 antibody. Following incubation the cells werestained for collagen using the Operetta assay and florescence wasquantified as described above. Secreted collagen levels in the cellculture supernatant were assessed by Sirius Red staining.

The results are shown in FIGS. 23A and 23B, and confirm theanti-fibrotic effect of inhibition of IL-11 using a neutralisingantibody.

Next, the ability of several other IL-11/IL-11R antagonists to inhibitfibrosis was analysed in vitro using the atrial fibroblast,TGFβ1-induced myofibroblast transition assay described herein above.

Briefly, human atrial fibroblasts cells were cultured in vitro,stimulated for 24 h with TGFβ1 (5 ng/ml) or left unstimulated, in thepresence/absence of: (i) neutralising anti-IL-11 antibody, (ii) aIL-11RA-gp130 fusion protein (iii) neutralising anti-IL-11RA antibody,(iv) treatment with siRNA directed against IL-11 or (v) treatment withsiRNA directed against IL-11RA. The proportion of activated fibroblasts(myofibroblasts) was analysed by evaluating αSMA content as describedabove.

The results are shown in FIG. 24. Each of the antagonists ofIL-11/IL-11R signaling was found to be able to abrogate TGFβ1-mediatedprofibrotic response.

Example 7 In Vivo Confirmation of a Profibrotic Role for IL-11/IL-11RSignaling

7.1 In Vitro Studies Using Cells Derived from IL-11RA Gene Knock-OutMice

All mice were bred and housed in the same room and provided food andwater ad libitum. Mice lacking functional alleles for IL-11Rα (IL-11RA1KO mice) were on C57Bl/6 genetic background. Mice were of 9-11 weeks ofage and the weight of animals did not differ significantly.

To further confirm the anti-fibrotic effect of inhibition ofIL-11/IL-11R signaling, primary fibroblasts were generated from IL-11RAgene knock-out mice and incubated with primary fibroblast cellsharvested from IL-11RA+/+ (i.e. wildtype), IL-11RA+/− (i.e. heterozygousknockout) and IL-11RA−/− (i.e. homozygous knockout) animals with TGFβ1,IL-11 or AngII. Activation and proliferation of fibroblasts and ECMproduction was analysed.

Fibroblasts derived from IL-11RA+/+, IL-11RA+/− and IL-11RA−/− mice wereincubated for 24 hours with TGFβ1, IL-11 or AngII (5 ng/ml). Followingincubation, cells were stained for αSMA content to estimate the fractionof myofibroblasts, for EdU to identify the fraction of proliferatingcells, and for collagen and periostin to estimate ECM production.Fluorescence was measured using the Operetta platform.

The results are shown in FIGS. 25A to 25D. IL-11RA−/− mice were foundnot to respond to pro-fibrotic stimuli. These results suggested thatIL-11 signaling is also required for AngII-induced fibrosis.

Next, it was investigated whether this was also true for otherpro-fibrotic cytokines. Briefly, fibroblasts were cultured in vitro inthe presence/absence of various different pro-fibrotic factors (ANG2,ET-1 or PDGF), and in the presence/absence of neutralising anti-IL-11antibody or pan anti-TGFβ antibody. After 24 hours, collagen productionby the cells was determined by analysis using the Operetta system asdescribed above, and myofibroblast generation was determined by analysisof αSMA expression as described above.

The results are shown in FIGS. 26A and 26B. IL-11 was found to berequired for fibrosis downstream of various profibrotic stimuli, and wasthus identified as a central mediator of fibrosis induced by a varietyof different profibrotic factors.

In a further experiment, the role of IL-11 signaling was investigated inlung fibrosis, using an in vitro scratch assay of migration of lungfibroblasts. In response to pro-fibrotic stimuli, fibroblasts areactivated and migrate within the fibrotic niche in the body. Themigration rate of cells is a measure of cell-cell and cell-matrixinteractions and a model for wound healing in vivo (Liang et al., 2007;Nat Protoc. 2(2):329-33).

Fibroblasts derived from lung tissue from both wild type (WT) and alsohomozygous IL-11RA (−/−) knockout mice were grown at low passage on aplastic surface until they formed a uniform cell monolayer. A scratchwas then created in the cell layer, and cell migration close to thescratch was monitored, either in the absence of stimulation, or in thepresence of TGFβ1 or IL-11. Images captured at images at the two timepoints of immediately after creating the scratch and at 24 h were usedto determine the area covered by cells, and the rate of migration wascompared between WT and KO fibroblasts. Cell migration (area in thescratch covered by cells after 24 h) was normalized to the migrationrate of WT cells without stimulus.

The results are shown in FIG. 40. Lung fibroblasts derived from WT micewere shown to migrate faster in the presence of TGFβ1 and IL-11,indicating a pro-fibrotic effect of both cytokines in lung fibroblasts.Cells lacking IL-11 signaling derived from KO mice migrated more slowlyas compared to WT cells. They also did not migrate faster in thepresence of TGFβ1. The scratch assay revealed that lung fibroblastslacking IL-11 signaling have a decrease cell migration rate both in thepresence of TGFβ1 or IL-11, and at baseline. Thus, inhibition of IL-11signaling is anti-fibrotic in the lung.

7.2 Heart Fibrosis

The efficacy of IL-11 inhibition to treat fibrotic disorders wasinvestigated in vivo. A mouse model for cardiac fibrosis, in whichfibrosis is induced by treatment with AngII, was used to investigatewhether IL-11RA −/− mice were protected from cardiac fibrosis.

Briefly, a pump was implanted, and wildtype (WT) IL-11RA(+/+) andknockout (KO) IL-11RA(−/−) mice were treated with AngII (2 mg/kg/day)for 28 days. At the end of the experiment, collagen content was assessedin the atria of the mice using a calorimetric hydroxyproline-based assaykit, and the level of RNA expression of the markers or fibrosis Col1A2,αSMA (ACTA2) and fibronectin (Fn1) were analysed by qPCR.

The results are shown in FIGS. 27A to 27D. The IL-11RA−/− mice werefound to be protected from the profibrotic effects of AngII.

7.3 Kidney Fibrosis

A mouse model for kidney fibrosis was established in wildtype (WT)IL-11RA(+/+) and knockout (KO) IL-11RA(−/−) mice by intraperitonealinjection of folic acid (180 mg/kg) in vehicle (0.3M NaHCO₃); controlmice were administered vehicle alone. Kidneys were removed 28 dayspost-injection, weighed and either fixed in 10% neutral-bufferedformalin for Masson's trichrome and Sirius staining or snap-frozen forcollagen assay, RNA, and protein studies.

Total RNA was extracted from the snap-frozen kidney using Trizol reagent(Invitrogen) and Qiagen TissueLyzer method followed by RNeasy column(Qiagen) purification. The cDNA was prepared using iScript™ cDNAsynthesis kit, in which each reaction contained 1 μg of total RNA, asper the manufacturer's instructions. Quantitative RT-PCR gene expressionanalysis was performed on triplicate samples with either TaqMan (AppliedBiosystems) or fast SYBR green (Qiagen) technology using StepOnePlus™(Applied Biosystem) over 40 cycles. Expression data were normalized toGAPDH mRNA expression level and we used the 2-ΔΔCt method to calculatethe fold-change. The snap-frozen kidneys were subjected to acidhydrolysis by heating in 6M HCl at a concentration of 50 mg/ml (95° C.,20 hours). The amount of total collagen in the hydrolysate wasquantified based on the colorimetric detection of hydroxyproline usingQuickzyme Total Collagen assay kit (Quickzyme Biosciences) as per themanufacturer's instructions.

The results of the analysis are shown in FIG. 28. Folate-induced kidneyfibrosis is shown to be dependent on IL-11 mediated signaling. Asignificant increase in collagen content in kidney tissue was observedin IL-11RA+/+ mice, indicative of kidney fibrosis. No significantincrease in collagen content was observed in IL-11RA −/− mice. Animalsdeficient for IL-11 signaling had significantly less collagen depositionin kidneys after toxic injury as compared to wild type animals.

7.4 Lung Fibrosis

IL-11 is confirmed as a key mediator of fibrosis in the lung, skin andeye in further in vivo models using the IL-11RA −/− knockout mice.Schematics of the experiments are shown in FIGS. 29A to 29C.

To analyse pulmonary fibrosis, IL-11RA −/− mice and IL-11RA +/+ mice aretreated by intratracheal administration of bleomycin on day 0 toestablish a fibrotic response in the lung (pulmonary fibrosis). Fibrosisof the lung develops by 21 days, at which point animals are sacrificedand analysed for differences in fibrosis markers between animals withand without IL-11 signaling. IL-11RA −/− mice have a reduced fibroticresponse in lung tissue as compared to IL-11RA +/+ mice, as evidenced byreduced expression of markers of fibrosis.

7.5 Skin Fibrosis

To analyse fibrosis of the skin, IL-11RA −/− mice and IL-11RA +/+ miceare treated by subcutaneous administration of bleomycin on day 0 toestablish a fibrotic response in the skin. Fibrosis of the skin developsby 28 days, at which point animals are sacrificed and analysed fordifferences in fibrosis markers between animals with and without IL-11signaling. IL-11RA −/− mice have a reduced fibrotic response in skintissue as compared to IL-11RA +/+ mice, as evidenced by reducedexpression of markers of fibrosis.

7.6 Eye Fibrosis

To analyse fibrosis in the eye, IL-11RA −/− mice and IL-11RA +/+ miceundergo trabeculectomy on day 0 to initiate a wound healing response inthe eye. Fibrosis of the eye develops within 7 days. The fibroticresponse is measured and compared between the IL-11RA −/− mice andIL-11RA +/+ mice. IL-11RA −/− mice have a reduced fibrotic response ineye tissue as compared to IL-11RA +/+ mice, as evidenced by reducedexpression of markers of fibrosis.

7.7 Other Tissues

The effect of IL-11RA knockout on fibrosis is also analysed in mousemodels of fibrosis for other tissues, such as the liver, bowel, and isalso analysed in a model relevant to multiorgan (i.e. systemic)fibrosis. The fibrotic response is measured and compared between theIL-11RA −/− mice and IL-11RA +/+ mice. IL-11RA −/− mice have a reducedfibrotic response as compared to IL-11RA +/+ mice, as evidenced byreduced expression of markers of fibrosis.

Example 8 Analysis of the Molecular Mechanisms Underlying IL-11-MediatedInduction of Fibrosis

The canonical mode of action of IL-11 is thought to be regulation of RNAexpression via STATS-mediated transcription (Zhu et al., 2015 PLoS ONE10, e0126296), and also through activation of ERK.

STAT3 activation is observed following stimulation with IL-11. However,when fibroblasts are incubated with TGFβ1, only activation of thecanonical SMAD pathway and ERK pathways is seen, and activation of STAT3is not observed, even in spite of the fact that IL-11 is secreted inresponse to TGFβ1. Only ERK activation is common to both TGFβ1 and IL-11signal transduction.

Cross-talk between TGFβ1 and IL-6 signaling has previously beendescribed, wherein TGFβ1 blocks the activation of STAT3 by IL-6 (Waliaet al., 2003 FASEB J. 17, 2130-2132). Given the close relationshipbetween IL-6 and IL-11, similar cross-talk may be observed for IL-11mediated signaling.

The inventors investigated by RNA-seq analysis whether regulation of RNAabundance was the underlying mechanism for the increased expression offibrosis marker proteins in response to IL-11, which would suggest STAT3as the underlying signaling pathway for IL-11 mediated profibroticprocesses. Fibroblasts were incubated for 24 hours either withoutstimulus, or in the presence of TGFβ1, IL-11 or TGFβ1 and IL-11.

The results are shown in FIG. 30A. TGFβ1 induced the expression ofcollagen, ACTA2 (αSMA) and other fibrosis marker at the RNA level.However, IL-11 did not regulate the expression of these genes, but adifferent set of genes.

Gene ontology analysis suggests that a pro-fibrotic effect infibroblasts is driven by IL-11-regulated RNA expression. Both TGFβ1 andIL-11 regulate an almost completely different set of genes on the RNAlevel.

Whilst TGFβ1 increases IL-11 secretion, the target genes of IL-11 arenot regulated when both TGFβ1 and IL-11 are present. This suggests thatTGFβ1 upregulates IL-11 and simultaneously blocks the canonicalIL-11-driven regulation of RNA expression via STAT3, similar to what isknown about the interaction of TGFβ1 and IL-6 pathways (Walia et al.,2003 FASEB J. 17, 2130-2132).

We also analysed whether RNA expression differences induced by TGFβ1 aredependent on IL-11 signaling, by analysing changes in RNA expression infibroblasts obtained from IL-11RA −/− mice as compared to IL-11RA +/+mice. RNA expression regulated by TGFβ1 is still observed when IL-11RAknockout cells were stimulated with TGFβ1, and RNA levels of αSMA,collagen etc. were still upregulated in the absence of IL-11 signaling(in IL-11RA −/− fibroblasts). When the pro-fibrotic effect of IL-11 andthe anti-fibrotic effect of IL-11 inhibition was investigated in vitro,reduced expression of markers of fibrosis was only observed at theprotein level, not at the transcriptional level as determined by qPCR.

The activation of non-canonical pathways (e.g. ERK signal transduction)is known to be crucial for the pro-fibrotic action of TGFβ1 (Guo andWang, 2008 Cell Res 19, 71-88). It is likely that non-canonical pathwaysare likely to be important for signaling for all known pro-fibroticcytokines, and that IL-11 is a post-transcriptional regulator which isessential for fibrosis.

Example 9 Human Anti-Human IL-11 Antibodies

Fully human anti-human IL-11 antibodies were developed via phagedisplay.

Recombinant human IL-11 (Cat. No. Z03108-1) and recombinant murine IL-11(Cat. No. Z03052-1) were obtained from GenScript (NJ, USA). Recombinanthuman IL-11 was expressed in CHO cells, both as an Fc-tagged version anda tag-free version. Tag-free murine IL-11 was expressed in HEK293 cells.

IL-11 bioactivity of recombinant human IL-11 and mouse IL-11 wasconfirmed by in vitro analysis using primary fibroblast cell cultures.

Recombinant, biotinylated human IL-11 and murine IL-11 were alsoprepared by biotinylation of the recombinant human IL-11 and murineIL-11 molecules, according to standard methods.

Antibodies capable of binding to both human IL-11 and murine IL-11 (i.e.cross-reactive antibodies) were identified by phage display using ahuman naïve library by panning using biotinylated and non-biotinylatedrecombinant human and murine IL-11, based on 16 different panningstrategies.

The phage display identified 175 scFv binders, as ‘first hits’. Sequenceanalysis of the CDR sequences from these 175 scFv identified 86 uniquescFv.

The soluble scFv were produced by recombinant expression in E. coli, andanalysed for their ability to bind to human IL-11 and murine IL-11 byELISA. Briefly, the respective antigen was coated to wells of an ELISAplate, the cell culture supernatant containing the respective scFv wasadded at a 1:2 dilution, and binding was detected.

The results of the ELISA analysis revealed:

-   -   8 scFV capable of binding only to human IL-11;    -   6 scFv capable of binding to murine IL-11 only;    -   32 scFv displaying only weak binding to human/murine IL-11, with        a high signal to noise ratio, and;    -   40 scFv having cross-reactivity for both human IL-11 and murine        IL-11.

From these 86 scFV, 56 candidates were selected for further functionalcharacterisation. For further analyses, the scFV were cloned intoscFV-Fc format in E. coli.

The VH and VL sequences of the antibodies were cloned into expressionvectors for the generation of scFv-Fc (human IgG1) antibodies. Thevectors were transiently expressed in mammalian cells cultured inserum-free media, and isolated by protein A purification.

Example 10 Functional Characterisation of Human Anti-Human IL-11Antibodies

The antibodies described in Example 9 were analysed in in vitro assaysfor their ability to (i) inhibit human IL-11-mediated signaling, and(ii) inhibit mouse IL-11-mediated signaling. The affinity of theantibodies for human IL-11 was also analysed by ELISA.

10.1 Ability to Inhibit Human IL-11 Mediated Signaling

To investigate ability to neutralise human IL-11-mediated signaling,cardiac atrial human fibroblasts were cultured in wells of 96-wellplates in the presence of TGFβ1 (5 ng/ml) for 24 hours, in the presenceor absence of the anti-IL-11 antibodies. TGFβ1 promotes the expressionof IL-11, which in turn drives the transistion of quiescent fibroblaststo activated, αSMA-positive fibroblasts. It has previously been shownthat neutralising IL-11 prevents TGFβ1-induced transition to activated,αSMA-positive fibroblasts.

Expression of αSMA was analysed with the Operetta High-Content ImagingSystem in an automated high-throughput fashion.

In non-stimulated cultures, ˜29.7% (=1) of the fibroblasts wereαSMA-positive, activated fibroblasts at the end of the 24 hour cultureperiod, whilst ˜52% (=1.81) of fibroblasts were αSMA-positive incultures that were stimulated with TGFβ1 in the absence of anti-IL-11antibodies.

Anti-IL-11 antibodies (2 μg/ml) were added to fibroblast cultures thatwere stimulated with TGFβ1, and at the end of the 24 hour cultureperiod, the percentage of αSMA-positive fibroblasts was determined. Thepercentages were normalised based on the percentage of αSMA-positivefibroblasts observed in cultures of fibroblasts which had not beenstimulated with TGFβ1.

28 of the antibodies were demonstrated to be capable of neutralisingsignaling mediated by human IL-11.

A commercial monoclonal mouse anti-IL-11 antibody (Monoclonal MouseIgG2A; Clone #22626; Catalog No. MAB218; R&D Systems, MN, USA) was alsoanalysed for ability to inhibit signaling by human IL-11 in theexperiments. This antibody was found to be able to reduce the percentageof activated fibroblasts to 28.3% (=0.99).

Several of the clones neutralised signaling by human IL-11 to a greaterextent than the commercially available mouse anti-IL-11 antibody(industry standard.

10.2 Ability to Inhibit Mouse IL-11 Mediated Signaling

The ability of the human antibodies to inhibit mouse IL-11-mediatedsignaling was also investigated, following the same procedure asdescribed in section 10.1 above, but using mouse dermal fibroblastsinstead of human atrial fibroblasts.

After 24 hours in culture, about 31.8% (=1) of non-stimulated cells inculture were activated fibroblasts. Stimulation with TGFβ1 resulted in a˜2-fold increase in the percentage of activated fibroblasts (68.8%=2.16)as compared to non-stimulated cultures.

The antibodies were demonstrated to be capable of neutralising signalingmediated by mouse IL-11. Monoclonal Mouse IgG2A clone #22626, catalogNo. MAB218 anti-IL-11 antibody was also analysed for ability to inhibitsignaling by mouse IL-11. This antibody was found to be able to reducethe percentage of activated fibroblasts to 39.4% (=1.24).

Several of the clones neutralised signaling by mouse IL-11 to a greaterextent than the commercially available mouse anti-IL-11 antibody(industry standard).

10.3 Analysis of Antibody Affinity for Human IL-11

The human anti-human IL-11 antibodies were analysed for their affinityof binding to human IL-11 by ELISA assay.

Recombinant human IL-11 was obtained from Genscript and Horseradishperoxidase (HRP)-conjugated anti-human IgG (Fc-specific) antibody wasobtained from Sigma. Corning 96-well ELISA plates were obtained fromSigma. Pierce 3,3′,5,5′-tetramethylbenzidine (TMB) ELISA substrate kitwas obtained from Life Technologies (0.4 g/mL TMB solution, 0.02%hydrogen peroxide in citric acid buffer). Bovine serum albumin andsulphuric acid was obtained from Sigma. Wash buffer comprised 0.05%Tween-20 in phosphate buffered saline (PBS-T). ScFv-Fc antibodies weregenerated as described in above. Purified mouse and human IgG controlswere purchased from Life Technologies. Tecan Infinite 200 PRO NanoQuantwas used to measure absorbance.

Criss-cross serial dilution analysis was performed as described byHornbeck et al., (2015) Curr Protoc Immunol 110, 2.1.1-23) to determinethe optimal concentration of coating antigen, primary and secondaryantibodies.

An indirect ELISA was performed to assess the binding affinity ofprimary ScFv-Fc antibodies at 50% of effective concentration (EC₅₀) aspreviously described (Unverdorben et al., (2016) MAbs 8, 120-128.).ELISA plates were coated with 1 μg/mL of recombinant human IL-11overnight at 4° C. and remaining binding sites were blocked with 2% BSAin PBS. ScFv-Fc antibodies were diluted in 1% BSA in PBS, titrated toobtain working concentrations of 800, 200, 50, 12.5, 3.125, 0.78, 0.195,and 0.049 ng/mL, and incubated in duplicates for 2 hours at roomtemperature. Detection of antigen-antibody binding was performed with15.625 ng/mL of HRP-conjugated anti-human IgG (Fc-specific) antibody.Following 2 hours of incubation with the detection antibody, 100 μl ofTMB substrate was added for 15 mins and chromogenic reaction stoppedwith 100 μl of 2 M H₂SO₄. Absorbance reading was measured at 450 nm withreference wavelength correction at 570 nm. Data were fitted with GraphPad Prism software with log transformation of antibody concentrationsfollowed by non-linear regression analysis with the asymmetrical(five-parameter) logistic dose-response curve to determine individualEC50 values.

The same materials and procedures as described above were performed todetermine the affinity of binding for the murine monoclonal anti-IL-11antibodies, with the exception that HRP-conjugated anti-mouse IgG (H&L)was used instead of HRP-conjugated anti-human IgG.

The same materials and procedures as described above were performed todetermine the affinity of binding for the human monoclonal anti-IL-11antibodies and murine monoclonal anti-IL-11 antibodies to recombinantmurine IL-11 obtained from Genscript.

The results of the ELISA assays were used to determine EC₅₀ values forthe antibodies.

10.4 Ability to Inhibit Human IL-11 Mediated Signaling in a Variety ofTissues

Ability of the antibodies to neutralise IL-11-mediated signaling infibroblasts obtained from a variety of different tissues isinvestigated, essentially as described in section 10.1 except thatinstead of cardiac atrial human fibroblasts, human fibroblasts derivedfrom liver, lung, kidney, eye, skin, pancreas, spleen, bowel, brain, andbone marrow are used for the experiments.

Anti-IL-11 antibodies are demonstrated to be capable of neutralisingsignaling in fibroblasts derived from the various different tissues, asdetermined by observation of a relative decrease in the proportion ofαSMA-positive fibroblasts at the end of the 24 h culture period in thepresence of the anti-IL-11 antibodies as compared to culture in theabsence of the antibodies.

Example 11 Inhibition of Fibrosis In Vivo Using Anti-IL-11 Antibodies

The therapeutic utility of the anti-human IL-11 antibodies isdemonstrated in in vivo mouse models of fibrosis for various differenttissues.

11.1 Heart Fibrosis

A pump is implanted, and mice are treated with AngII (2 mg/kg/day) for28 days.

Neutralising anti-IL-11 antibodies, or control antibodies, areadministered to different groups of mice by intravenous injection. Atthe end of the experiment, collagen content is assessed in the atria ofthe mice using a calorimetric hydroxyproline-based assay kit, and thelevel of RNA expression of the markers or fibrosis Col1A2, αSMA (ACTA2)and fibronectin (Fn1) were analysed by qPCR.

Mice treated with neutralising anti-IL-11 antibodies have a reducedfibrotic response in heart tissue as compared to mice treated withcontrol antibodies, as evidenced by reduced expression of markers offibrosis.

11.2 Kidney Fibrosis

A mouse model for kidney fibrosis is established, in which fibrosis isinduced by intraperitoneal injection of folic acid (180 mg/kg) invehicle (0.3M NaHCO₃); control mice were administered vehicle alone.

Neutralising anti-IL-11 antibodies, or control antibodies, areadministered to different groups of mice by intravenous injection.Kidneys are removed at day 28, weighed and either fixed in 10%neutral-buffered formalin for Masson's trichrome and Sirius staining orsnap-frozen for collagen assay, RNA, and protein studies.

Total RNA is extracted from the snap-frozen kidney using Trizol reagent(Invitrogen) and Qiagen TissueLyzer method followed by RNeasy column(Qiagen) purification. The cDNA is prepared using iScript™ cDNAsynthesis kit, in which each reaction contained 1 μg of total RNA, asper the manufacturer's instructions. Quantitative RT-PCR gene expressionanalysis is performed on triplicate samples with either TaqMan (AppliedBiosystems) or fast SYBR green (Qiagen) technology using StepOnePlus™(Applied Biosystem) over 40 cycles. Expression data are normalized toGAPDH mRNA expression level and the 2-ΔΔCt method is used to calculatethe fold-change. The snap-frozen kidneys are subjected to acidhydrolysis by heating in 6M HCl at a concentration of 50 mg/ml (95° C.,20 hours). The amount of total collagen in the hydrolysate is quantifiedbased on the colorimetric detection of hydroxyproline using QuickzymeTotal Collagen assay kit (Quickzyme Biosciences) as per themanufacturer's instructions.

Mice treated with neutralising anti-IL-11 antibodies have a reducedfibrotic response in kidney tissue as compared to mice treated withcontrol antibodies, as evidenced by reduced expression of markers offibrosis.

11.3 Lung Fibrosis

Mice are treated by intratracheal administration of bleomycin on day 0to establish a fibrotic response in the lung (pulmonary fibrosis).

Neutralising anti-IL-11 antibodies, or control antibodies, areadministered to different groups of mice by intravenous injection. Miceare sacrificed at day 21, and analysed for differences in fibrosismarkers.

Mice treated with neutralising anti-IL-11 antibodies have a reducedfibrotic response in lung tissue as compared to mice treated withcontrol antibodies, as evidenced by reduced expression of markers offibrosis.

11.4 Skin Fibrosis

Mice are treated by subcutaneous administration of bleomycin on day 0 toestablish a fibrotic response in the skin.

Neutralising anti-IL-11 antibodies, or control antibodies, areadministered to different groups of mice by intravenous injection. Miceare sacrificed at day 21, and analysed for differences in fibrosismarkers.

Mice treated with neutralising anti-IL-11 antibodies have a reducedfibrotic response in skin tissue as compared to mice treated withcontrol antibodies, as evidenced by reduced expression of markers offibrosis.

11.5 Eye Fibrosis

Mice undergo trabeculectomy on day 0 to initiate a wound healingresponse in the eye.

Neutralising anti-IL-11 antibodies, or control antibodies, areadministered to different groups of mice by intravenous injection, andfibrosis is monitored in the eye tissue.

Mice treated with neutralising anti-IL-11 antibodies have a reducedfibrotic response in eye tissue as compared to mice treated with controlantibodies, as evidenced by reduced expression of markers of fibrosis.

11.6 Other Tissues

The effect of treatment with neutralising anti-IL-11 antibodies onfibrosis is also analysed in mouse models of fibrosis for other tissues,such as the liver, kidney, bowel, and is also analysed in a modelrelevant to multiorgan (i.e. systemic) fibrosis.

Mice treated with neutralising anti-IL-11 antibodies have a reducedfibrotic response as compared to mice treated with control antibodies,as evidenced by reduced expression of markers of fibrosis.

Example 12 Antihuman IL-11Rα Antibodies

Mouse monoclonal antibodies directed against human IL-11Rα protein weregenerated as follows.

cDNA encoding the amino acid for human IL-11Rα was cloned intoexpression plasmids (Aldevron GmbH, Freiburg, Germany).

Mice were immunised by intradermal application of DNA-coatedgold-particles using a hand-held device for particle-bombardment (“genegun”). Serum samples were collected from mice after a series ofimmunisations, and tested in flow cytometry on HEK cells which had beentransiently transfected with human IL-11Rα expression plasmids (cellsurface expression of human IL-11Rα by transiently transfected HEK cellswas confirmed with anti-tag antibodies recognising a tag added to theN-terminus of the IL-11Rα protein).

Antibody-producing cells were isolated from the mice and fused withmouse myeloma cells (Ag8) according to standard procedures.

Hybridomas producing antibodies specific for IL-11Rα were identified byscreening for ability to bind to IL-11Rα expressing HEK cells by flowcytometry.

Cell pellets of positive hybridoma cells were prepared using an RNAprotection agent (RNAlater, cat. #AM7020 by ThermoFisher Scientific) andfurther processed for sequencing of the variable domains of theantibodies.

Sequencing was performed using Big Dye® Terminator v3.1 Cycle Sequencingkit (Life Technologies®) according to the manufacturer's instructions.All data was collected using a 3730xl DNA Analyzer system and UnifiedData Collection software (Life Technologies®). Sequence assembly wasperformed using CodonCode Aligner (CodonCode Corporation). Mixed basecalls were resolved by automatically assigning the most prevalent basecall to the mixed base calls. Prevalence was determined by bothfrequency of a base call and the individual quality of the base calls.

In total, 17 mouse monoclonal anti-human IL-11Rα antibody clones weregenerated.

Example 13 Functional Characterisation of Anti-Human IL-11Rα Antibodies

13.1 Ability to Inhibit Human IL-11/IL-11R Mediated Signaling

To investigate the ability of the anti-IL-11Rα antibodies to neutralisehuman IL-11/IL-11R mediated signaling, cardiac atrial human fibroblastswere cultured in wells of 96-well plates in the presence of TGFβ1 (5ng/ml) for 24 hours, in the presence or absence of the anti-IL-11Rαantibodies. This profibrotic stimulus promotes the expression of IL-11,which in turn drives the transistion of quiescent fibroblasts toactivated, αSMA-positive fibroblasts. It has previously been shown thatneutralising IL-11 prevents TGFβ1-induced transition to activated,αSMA-positive fibroblasts.

Anti-IL-11Rα antibodies (2 μg/ml) were added to fibroblast cultures thatwere stimulated with TGFβ1, and at the end of the 24 hour cultureperiod, the percentage of αSMA-positive fibroblasts was determined. Thepercentages were normalised based on the percentage of αSMA-positivefibroblasts observed in cultures of fibroblasts which had not beenstimulated with TGFβ1.

Expression of αSMA was analysed with the Operetta High-Content ImagingSystem in an automated high-throughput fashion.

Stimulation with TGFβ1 resulted in a 1.58 fold increase in the number ofαSMA-positive, activated fibroblasts at the end of the 24 hour cultureperiod in the absence of anti-IL-11Rα antibodies.

A commercial monoclonal mouse anti-IL-11 antibody (Monoclonal MouseIgG2A; Clone #22626; Catalog No. MAB218; R&D Systems, MN, USA) wasincluded as a control. This antibody was found to be able to reduce thepercentage of activated fibroblasts to 0.89 fold of the percentage ofactivated fibroblasts in unstimulated cultures (i.e. in the absence ofstimulation with TGFβ1).

The anti-IL-11Rα antibodies were found to be able to inhibitIL-11/IL-11R signaling in human fibroblasts, and several were able toinhibit IL-11/IL-11R signaling to a greater extent than the monoclonalmouse anti-IL-11 antibody.

13.2 Ability to Inhibit Mouse IL-11 Mediated Signaling

The ability of the anti-IL-11Rα antibodies to inhibit mouseIL-11-mediated signaling was also investigated, following the sameprocedure as described in section 13.1 above, but using mouse atrialfibroblasts instead of human atrial fibroblasts.

Stimulation with TGFβ1 resulted in a 2.24 fold increase in the number ofαSMA-positive, activated fibroblasts at the end of the 24 hour cultureperiod in the absence of anti-IL-11Rα antibodies.

The commercial monoclonal mouse anti-IL-11 antibody (Monoclonal MouseIgG2A; Clone #22626; Catalog No. MAB218; R&D Systems, MN, USA) wasincluded as a control. This antibody was found to be able to reduce thepercentage of activated fibroblasts to 1.44 fold of the percentage ofactivated fibroblasts in unstimulated cultures (i.e. in the absence ofstimulation with TGFβ1).

The anti-IL-11Rα antibodies were found to be able to inhibitIL-11/IL-11R signaling in mouse fibroblasts, and several were able toinhibit IL-11/IL-11R signaling to a greater extent than the monoclonalmouse anti-IL-11 antibody.

13.3 Screening for Ability to Bind IL-11Rα

The mouse hybridomas producing anti-human IL-11Rα antibodies weresub-cloned, and cell culture supernatant from the subcloned hybridomaswas analysed by “mix-and-measure” iQue assay for (i) ability to bind tohuman IL-11Rα, and (ii) cross reactivity for antigen other than IL-11Rα.

Briefly, labelled control cells (not expressing IL-11Rα at the cellsurface) and unlabelled target cells expressing human IL-11Rα at theirsurface (following transient transfection with a plasmid encoding aFLAG-tagged human IL-11Rα) were mixed together with the cell culturesupernatant (containing mouse-anti-IL-11Rα antibodies) and secondarydetection antibodies (fluorescently-labelled anti-mouse IgG antibody).

The cells were then analysed using the HTFC Screening System (iQue) forthe two labels (i.e. the cell label and the label on the secondaryantibody). Detection of the secondary antibody on the unlabelled,IL-11Rα expressing cells indicated ability of the mouse-anti-IL-11Rαantibodies to bind to IL-11Rα. Detection of the secondary antibody onthe labelled, control cells indicated cross-reactivity of themouse-anti-IL-11Rα antibodies for target other than IL-11Rα.

As a positive control condition, labelled and unlabelled cells wereincubated with a mouse anti-FLAG tag antibody as the primary antibody.

The majority of the subcloned hybridomas expressed antibody which wasable to bind to human IL-11Rα, and which recognised this target withhigh specificity.

13.4 Analysis of Antibody Affinity for Human IL-11Rα

The anti-human IL-11Rα antibodies are analysed for their affinity ofbinding to human IL-11Rα by ELISA assay.

Recombinant human IL-11Rα is obtained from Genscript and Horseradishperoxidase (HRP)-conjugated anti-human IgG (Fc-specific) antibody isobtained from Sigma. Corning 96-well ELISA plates are obtained fromSigma. Pierce 3,3′,5,5′-tetramethylbenzidine (TMB) ELISA substrate kitis obtained from Life Technologies (0.4 g/mL TMB solution, 0.02%hydrogen peroxide in citric acid buffer). Bovine serum albumin andsulphuric acid is obtained from Sigma. Wash buffer comprises 0.05%Tween-20 in phosphate buffered saline (PBS-T). Purified IgG controls arepurchased from Life Technologies. Tecan Infinite 200 PRO NanoQuant isused to measure absorbance.

Criss-cross serial dilution analysis was performed as described byHornbeck et al., (2015) Curr Protoc Immunol 110, 2.1.1-23) to determinethe optimal concentration of coating antigen, primary and secondaryantibodies.

An indirect ELISA is performed to assess the binding affinity of themouse anti-IL-11Rα antibodies at 50% of effective concentration (EC₅₀)as previously described (Unverdorben et al., (2016) MAbs 8, 120-128.).ELISA plates are coated with 1 μg/mL of recombinant human IL-11Rαovernight at 4° C., and remaining binding sites are blocked with 2% BSAin PBS. The antibodies are diluted in 1% BSA in PBS, titrated to obtainworking concentrations of 800, 200, 50, 12.5, 3.125, 0.78, 0.195, and0.049 ng/mL, and incubated in duplicates for 2 hours at roomtemperature. Detection of antigen-antibody binding is performed with15.625 ng/mL of HRP-conjugated anti-mouse IgG antibody. Following 2hours of incubation with the detection antibody, 100 μl of TMB substrateis added for 15 mins and chromogenic reaction stopped with 100 μl of 2 MH₂O₄. Absorbance reading is measured at 450 nm with reference wavelengthcorrection at 570 nm. Data are fitted with GraphPad Prism software withlog transformation of antibody concentrations followed by non-linearregression analysis with the asymmetrical (five-parameter) logisticdose-response curve to determine individual EC50 values.

13.5 Ability to Inhibit Human IL-11/IL-11R Signaling in a Variety ofTissues

Ability of the antibodies to neutralise IL-11/IL-11R signaling infibroblasts obtained from a variety of different tissues isinvestigated, essentially as described in section 13.1 except thatinstead of cardiac atrial human fibroblasts, human fibroblasts derivedfrom liver, lung, kidney, eye, skin, pancreas, spleen, bowel, brain, andbone marrow are used for the experiments.

Anti-IL-11Rα antibodies are demonstrated to be capable of neutralisingIL-11/IL-11R signaling in fibroblasts derived from the various differenttissues, as determined by observation of a relative decrease in theproportion of αSMA-positive fibroblasts at the end of the 24 h cultureperiod in the presence of the anti-IL-11Rα antibodies as compared toculture in the absence of the antibodies.

Example 14 Inhibition of Fibrosis In Vivo Using Anti-IL-11Rα Antibodies

The therapeutic utility of the anti-human IL-11Rα antibodies isdemonstrated in vivo in mouse models of fibrosis for various differenttissues.

14.1 Heart Fibrosis

A pump is implanted, and mice are treated with AngII (2 mg/kg/day) for28 days.

Neutralising anti-IL-11Rα antibodies, or control antibodies, areadministered to different groups of mice by intravenous injection. Atthe end of the experiment, collagen content is assessed in the atria ofthe mice using a calorimetric hydroxyproline-based assay kit, and thelevel of RNA expression of the markers or fibrosis Col1A2, αSMA (ACTA2)and fibronectin (Fn1) were analysed by qPCR.

Mice treated with neutralising anti-IL-11Rα antibodies have a reducedfibrotic response in heart tissue as compared to mice treated withcontrol antibodies, as evidenced by reduced expression of markers offibrosis.

14.2 Kidney Fibrosis

A mouse model for kidney fibrosis is established, in which fibrosis isinduced by intraperitoneal injection of folic acid (180 mg/kg) invehicle (0.3M NaHCO₃); control mice were administered vehicle alone.

Neutralising anti-IL-11Rα antibodies, or control antibodies, areadministered to different groups of mice by intravenous injection.Kidneys are removed at day 28, weighed and either fixed in 10%neutral-buffered formalin for Masson's trichrome and Sirius staining orsnap-frozen for collagen assay, RNA, and protein studies.

Total RNA is extracted from the snap-frozen kidney using Trizol reagent(Invitrogen) and Qiagen TissueLyzer method followed by RNeasy column(Qiagen) purification. The cDNA is prepared using iScript™ cDNAsynthesis kit, in which each reaction contained 1 μg of total RNA, asper the manufacturer's instructions. Quantitative RT-PCR gene expressionanalysis is performed on triplicate samples with either TaqMan (AppliedBiosystems) or fast SYBR green (Qiagen) technology using StepOnePlus™(Applied Biosystem) over 40 cycles. Expression data are normalized toGAPDH mRNA expression level and the 2-ΔΔCt method is used to calculatethe fold-change. The snap-frozen kidneys are subjected to acidhydrolysis by heating in 6M HCl at a concentration of 50 mg/ml (95° C.,20 hours). The amount of total collagen in the hydrolysate is quantifiedbased on the colorimetric detection of hydroxyproline using QuickzymeTotal Collagen assay kit (Quickzyme Biosciences) as per themanufacturer's instructions.

Mice treated with neutralising anti-IL-11Rα antibodies have a reducedfibrotic response in kidney tissue as compared to mice treated withcontrol antibodies, as evidenced by reduced expression of markers offibrosis.

14.3 Lung Fibrosis

Mice are treated by intratracheal administration of bleomycin on day 0to establish a fibrotic response in the lung (pulmonary fibrosis).

Neutralising anti-IL-11Rα antibodies, or control antibodies, areadministered to different groups of mice by intravenous injection. Miceare sacrificed at day 21, and analysed for differences in fibrosismarkers.

Mice treated with neutralising anti-IL-11Rα antibodies have a reducedfibrotic response in lung tissue as compared to mice treated withcontrol antibodies, as evidenced by reduced expression of markers offibrosis.

14.4 Skin Fibrosis

Mice are treated by subcutaneous administration of bleomycin on day 0 toestablish a fibrotic response in the skin.

Neutralising anti-IL-11Rα antibodies, or control antibodies, areadministered to different groups of mice by intravenous injection. Miceare sacrificed at day 21, and analysed for differences in fibrosismarkers.

Mice treated with neutralising anti-IL-11Rα antibodies have a reducedfibrotic response in skin tissue as compared to mice treated withcontrol antibodies, as evidenced by reduced expression of markers offibrosis.

14.5 Eye Fibrosis

Mice undergo trabeculectomy on day 0 to initiate a wound healingresponse in the eye.

Neutralising anti-IL-11Rα antibodies, or control antibodies, areadministered to different groups of mice by intravenous injection, andfibrosis is monitored in the eye tissue.

Mice treated with neutralising anti-IL-11Rα antibodies have a reducedfibrotic response in eye tissue as compared to mice treated with controlantibodies, as evidenced by reduced expression of markers of fibrosis.

14.6 Other Tissues

The effect of treatment with neutralising anti-IL-11Rα antibodies onfibrosis is also analysed in mouse models of fibrosis for other tissues,such as the liver, kidney, bowel, and is also analysed in a modelrelevant to multiorgan (i.e. systemic) fibrosis.

Mice treated with neutralising anti-IL-11Rα antibodies have a reducedfibrotic response as compared to mice treated with control antibodies,as evidenced by reduced expression of markers of fibrosis.

Example 15 Decoy IL-11 Receptors

15.1 Decoy IL-11 Receptor Constructs

Decoy IL-11 Receptor molecules were designed and cloned into the pTT5vector for recombinant expression in 293-6E cells.

Briefly, an insert for the plasmid comprising cDNA encoding the ligandbinding domains D1, D2 and D3 of gp130 in-frame with cDNA encodingeither a 50 amino acid or 33 amino acid linker region, followed by cDNAencoding the ligand binding domains D2 and D3 of human IL-11Rα, followedby cDNA encoding the FLAG tag. The cDNA insert incorporated a leadersequence, Kozak sequences at the 5′ end, and included a 5′ EcoRIrestriction site and a 3′ HindIII restriction site (downstream of a stopcodon) for insertion into the pTT5 vector.

The two constructs encoding a decoy IL-11 receptor molecule havingeither a 50 amino acid or 33 amino acid sequence are respectivelydesignated Decoy IL-11 Receptor 1 (D11R1) and Decoy IL-11 Receptor 2(D11R2).

15.2 Decoy IL-11 Receptor Expression and Purification

The constructs were transfected into 293-6E cells for recombinantexpression and purification.

293-6E cells were grown in serum-free FreeStyle™ 293 Expression Medium(Life Technologies, Carlsbad, Calif., USA). Cells were maintained inErlenmeyer Flasks (Corning Inc., Acton, Mass.) at 37° C. with 5% CO₂ onan orbital shaker (VWR Scientific, Chester, Pa.).

One day before transfection, the cells were seeded at an appropriatedensity in Corning Erlenmeyer Flasks. On the day of transfection, DNAand transfection reagent were mixed at an optimal ratio and then addedinto the flask with cells ready for transfection. The recombinantplasmids encoding D11R1 and D11R2 were transiently transfected intosuspension 293-6E cell cultures on two separate days.

Cell culture supernatants were collected on day 6 and used forpurification. Briefly, cell culture broths were centrifuged andfiltrated. 0.5 ml of resin was added to cell culture supernatants andincubated for 3-4 hours to capture the target protein.

After washing and elution with appropriate buffers, eluted fractionswere analysed by SDS-PAGE and Western blot using Rabbit anti-FLAGpolyclonal Ab (GenScript, Cat. No. A00170) to confirm expression of theFLAG-tagged decoy IL-11 receptor molecules.

The purified species were quantified and stored at −80° C.

Example 16 Functional Characterisation of Decoy IL-11 Receptors

16.1 Ability to Inhibit Human IL-11 Mediated Signaling

To investigate ability to neutralise human IL-11-mediated signaling,cardiac atrial human fibroblasts were cultured in wells of 96-wellplates in the presence of TGFβ1 (5 ng/ml) for 24 hours, in the presenceor absence of various concentrations of D11R1 or D11R2.

TGFβ1 promotes the expression of IL-11, which in turn drives thetransition of quiescent fibroblasts to activated, αSMA-positivefibroblasts. It has previously been shown that neutralising IL-11prevents TGFβ1-induced transition to activated, αSMA-positivefibroblasts.

Expression of αSMA was analysed with the Operetta High-Content ImagingSystem in an automated high-throughput fashion.

D11R1 or D11R2 were added to fibroblast cultures that were stimulatedwith TGFβ1 at final concentrations of 5 ng/ml, 50 ng/ml and 500 ng/ml,and at the end of the 24 hour culture period, the percentage ofαSMA-positive fibroblasts in the culture was determined.

Both D11R1 and D11R2 were demonstrated to be capable of neutralisingsignaling mediated by human IL-11 in a dose-dependent manner.

The results of the experiments are shown in FIGS. 32A and 32B. BothD11R1 and D11R2 were demonstrated to be capable of neutralisingsignaling mediated by human IL-11 in a dose-dependent manner.

The IC₅₀ for the D11R1 and D11R2 molecules was determined to be ˜1 nM.

16.2 Ability to Inhibit Mouse IL-11 Mediated Signaling

The ability of D11R1 and D11R2 to inhibit mouse IL-11-mediated signalingis investigated, following the same procedure as described in section16.1 above, but using mouse dermal fibroblasts instead of human atrialfibroblasts.

D11R1 and D11R2 are demonstrated to be capable of neutralisingIL-11/IL-11R signaling in mouse dermal fibroblasts, as determined byobservation of a relative decrease in the proportion of αSMA-positivefibroblasts at the end of the 24 h culture period in the presence ofD11R1 or D11R2 as compared to culture in the absence of the decoy IL-11receptors.

16.3 Analysis of Decoy IL-11 Receptor Affinity for IL-11

D11R1 and D11R2 are analysed for their affinity of binding to humanIL-11 by ELISA assay.

Recombinant human IL-11 was obtained from Genscript and Horseradishperoxidase (HRP)-conjugated anti-FLAG antibody is obtained. Corning96-well ELISA plates were obtained from Sigma. Pierce3,3′,5,5′-tetramethylbenzidine (TMB) ELISA substrate kit was obtainedfrom Life Technologies (0.4 g/mL TMB solution, 0.02% hydrogen peroxidein citric acid buffer). Bovine serum albumin and sulphuric acid wasobtained from Sigma. Wash buffer comprised 0.05% Tween-20 in phosphatebuffered saline (PBS-T). Tecan Infinite 200 PRO NanoQuant is used tomeasure absorbance.

An indirect ELISA is performed to assess the binding affinity of D11R1and D11R2 at 50% of effective concentration (EC₅₀) as previouslydescribed (Unverdorben et al., (2016) MAbs 8, 120-128.). ELISA platesare coated with 1 μg/mL of recombinant human IL-11 overnight at 4° C.and remaining binding sites were blocked with 2% BSA in PBS. D11R1 andD11R1 are diluted in 1% BSA in PBS, titrated to obtain workingconcentrations of 800, 200, 50, 12.5, 3.125, 0.78, 0.195, and 0.049ng/mL, and incubated in duplicates for 2 hours at room temperature.Detection of antigen-decoy IL-11 receptor binding is performed withHRP-conjugated anti-FLAG antibody. Following 2 hours of incubation withthe detection antibody, 100 μl of TMB substrate is added for 15 mins andchromogenic reaction stopped with 100 μl of 2 M H₂SO₄. Absorbancereading is measured at 450 nm with reference wavelength correction at570 nm. Data are fitted with Graph Pad Prism software with logtransformation of decoy IL-11 receptor concentrations followed bynon-linear regression analysis with the asymmetrical (five-parameter)logistic dose-response curve to determine EC50 values.

The same materials and procedures as described above were performed todetermine the affinity of binding to recombinant murine IL-11 obtainedfrom Genscript.

16.4 Ability to Inhibit Human IL-11 Mediated Signaling in a Variety ofTissues

Ability of the decoy IL-11 receptors D11R1 and D11R2 to neutraliseIL-11-mediated signaling in fibroblasts obtained from a variety ofdifferent tissues is investigated, essentially as described in sections18.1 except that instead of cardiac atrial human fibroblasts, humanfibroblasts derived from liver, lung, kidney, eye, skin, pancreas,spleen, bowel, brain, and bone marrow are used for the experiments.

D11R1 and D11R2 are demonstrated to be capable of neutralising signalingin fibroblasts derived from the various different tissues, as determinedby observation of a relative decrease in the proportion of αSMA-positivefibroblasts at the end of the 24 h culture period in the presence of thedecoy IL-11 receptors as compared to culture in the absence of the decoyIL-11 receptors.

Example 17 Inhibition of Fibrosis In Vivo Using Decoy IL-11 Receptors

The therapeutic utility of the decoy IL-11 receptors is demonstrated inin vivo mouse models of fibrosis for various different tissues.

17.1 Heart Fibrosis

A pump is implanted, and mice are treated with AngII (2 mg/kg/day) for28 days.

Decoy IL-11 receptors D11R1 or D11R2 are administered to differentgroups of mice by intravenous injection. At the end of the experiment,collagen content is assessed in the atria of the mice using acalorimetric hydroxyproline-based assay kit, and the level of RNAexpression of the markers or fibrosis Col1A2, αSMA (ACTA2) andfibronectin (Fn1) were analysed by qPCR.

Mice treated with decoy IL-11 receptors have a reduced fibrotic responsein heart tissue as compared to untreated/vehicle treated controls, asevidenced by reduced expression of markers of fibrosis.

17.2 Kidney Fibrosis

A mouse model for kidney fibrosis is established, in which fibrosis isinduced by intraperitoneal injection of folic acid (180 mg/kg) invehicle (0.3M NaHCO₃); control mice were administered vehicle alone.

Decoy IL-11 receptors D11R1 or D11R2 are administered to differentgroups of mice by intravenous injection. Kidneys are removed at day 28,weighed and either fixed in 10% neutral-buffered formalin for Masson'strichrome and Sirius staining or snap-frozen for collagen assay, RNA,and protein studies.

Total RNA is extracted from the snap-frozen kidney using Trizol reagent(Invitrogen) and Qiagen TissueLyzer method followed by RNeasy column(Qiagen) purification. The cDNA is prepared using iScript™ cDNAsynthesis kit, in which each reaction contained 1 μg of total RNA, asper the manufacturer's instructions. Quantitative RT-PCR gene expressionanalysis is performed on triplicate samples with either TaqMan (AppliedBiosystems) or fast SYBR green (Qiagen) technology using StepOnePlus™(Applied Biosystem) over 40 cycles. Expression data are normalized toGAPDH mRNA expression level and the 2-ΔΔCt method is used to calculatethe fold-change. The snap-frozen kidneys are subjected to acidhydrolysis by heating in 6M HCl at a concentration of 50 mg/ml (95° C.,20 hours). The amount of total collagen in the hydrolysate is quantifiedbased on the colorimetric detection of hydroxyproline using QuickzymeTotal Collagen assay kit (Quickzyme Biosciences) as per themanufacturer's instructions.

Mice treated with decoy IL-11 receptors have a reduced fibrotic responsein kidney tissue as compared to untreated/vehicle treated controls, asevidenced by reduced expression of markers of fibrosis.

17.3 Lung Fibrosis

Mice are treated by intratracheal administration of bleomycin on day 0to establish a fibrotic response in the lung (pulmonary fibrosis).

Decoy IL-11 receptors D11R1 or D11R2 are administered to differentgroups of mice by intravenous injection. Mice are sacrificed at day 21,and analysed for differences in fibrosis markers.

Mice treated with decoy IL-11 receptors have a reduced fibrotic responsein lung tissue as compared to untreated/vehicle treated controls, asevidenced by reduced expression of markers of fibrosis.

17.4 Skin Fibrosis

Mice are treated by subcutaneous administration of bleomycin on day 0 toestablish a fibrotic response in the skin.

Decoy IL-11 receptors D11R1 or D11R2 are administered to differentgroups of mice by intravenous injection. Mice are sacrificed at day 21,and analysed for differences in fibrosis markers.

Mice treated with decoy IL-11 receptors have a reduced fibrotic responsein skin tissue as compared to untreated/vehicle treated controls, asevidenced by reduced expression of markers of fibrosis.

17.5 Eye Fibrosis

Mice undergo trabeculectomy procedure as described in Example 7.6 aboveto initiate a wound healing response in the eye.

Decoy IL-11 receptors D11R1 or D11R2 are administered to differentgroups of mice by intravenous injection, and fibrosis is monitored inthe eye tissue.

Mice treated with decoy IL-11 receptors have a reduced fibrotic responsein eye tissue as compared to untreated/vehicle treated controls, asevidenced by reduced expression of markers of fibrosis.

17.6 Other Tissues

The effect of treatment with decoy IL-11 receptors D11R1 or D11R2 onfibrosis is also analysed in mouse models of fibrosis for other tissues,such as the liver, kidney, bowel, and is also analysed in a modelrelevant to multiorgan (i.e. systemic) fibrosis.

The fibrotic response is measured and compared between mice treated withdecoy IL-11 receptors and untreated mice, or vehicle treated controls.Mice treated with decoy IL-11 receptors have a reduced fibrotic responseas compared to untreated/vehicle treated controls, as evidenced byreduced expression of markers of fibrosis.

Example 18 Genetic Biomarkers for IL-11 Response

In addition to measuring IL-11 protein as a potential biomarker forfibrosis, we developed an assay that can predict IL-11 secretion statusin humans. This assay could be used as a companion diagnostic inIL-11-related clinical trials.

We first generated RNA-seq data (FIG. 16) and determined the genotype of69 ethnically matched (Chinese) individuals in the cohort using a SNParray based on fluorescent probe hybridization supplied by Illumina(HumanOmniExpress 24).

We then performed genome-wide linkage eQTL analysis to assess whetherSingle Nucleotide Polymorphisms (SNPs) affect RNA transcript levels ofIL-11 or IL-11RA in unstimulated fibroblasts, in TGFB1 stimulated (5ng/ml, 24 h) fibroblasts. We also tested if the increase in IL-11 uponTGFβ1 stimulation (=response) was dependent on the genotype.

At first we quantified the read count for both IL-11 and IL-11RA in allindividuals and transformed these counts using the variancestabilization (VST) approach of the DESeq2 method (Love et al., GenomeBiology 2014 15:550). We then considered IL-11 and IL-11RA expression inunstimulated (VST_(unstim)) and stimulated (VST_(stim)) cells. To assessthe increase in IL-11, we also computed the delta in expression asVST_(stim)−VST_(unstim). We corrected the expression values usingcovariates such as RNA sequencing library batch, RNA RIN quality score,library concentration, library fragment size, age, gender beforeanalyses. SNP and transcript expression, or delta expression, pairs wereanalysed using the matrix eQTL approach (Andrey A. Shabalin.,Bioinformatics 2012 May 15; 28(10): 1353-1358).

We did not observe variation in cis or trans that significantly affectedIL-11 expression in unstimulated cells. However, we detected distantSNPs that regulated the expression in stimulated=fibrotic fibroblasts.These variants stratify the population between individuals that doexpress low levels of IL-11 and those that express high amounts of IL-11in fibrosis. We also detected local and distal variants that predictedthe increase in IL-11 expression in response to TGFβ1. These variantscan be used to stratify individuals into high and low responders infibrosis.

The SNPs identified are shown in FIGS. 33 to 35 and accompanying data isshown in FIGS. 36 and 37.

We claim:
 1. A method of treating a disease or condition characterisedby fibrosis of the eye in a human subject, the method comprisingadministering to the human subject in need of treatment atherapeutically effective amount of an Interleukin 11 receptor α(IL-11Rα) antibody which is capable of inhibiting Interleukin 11 (IL-11)mediated signaling.
 2. The method according to claim 1, wherein thedisease or condition is selected from the group consisting of Grave'sopthalmopathy, epiretinal fibrosis, retinal fibrosis, subretinalfibrosis, subretinal fibrosis associated with macular degeneration(AMD), subretinal fibrosis associated with wet AMD, diabeticretinopathy, glaucoma, corneal fibrosis, post-surgical fibrosis,fibrosis of the posterior capsule following cataract surgery, fibrosisof the bleb following trabeculectomy for glaucoma, conjunctival fibrosisand subconjunctival fibrosis.
 3. The method according to claim 1,wherein the antibody is capable of inhibiting or reducing the binding ofIL-11 to an IL-11 receptor.
 4. The method according to claim 1, whereinthe method comprises administering said antibody to a subject in whichIL-11 or IL-11R expression is upregulated.
 5. The method according toclaim 1, wherein the method comprises administering said antibody to asubject in which IL-11 or IL-11R expression has been determined to beupregulated.
 6. The method according to claim 1, wherein the methodcomprises determining whether IL-11 or IL-11R expression is upregulatedin the subject and administering said antibody to a subject in whichIL-11 or IL-11R expression is upregulated.
 7. A method of treating adisease or condition characterized by fibrosis selected from the groupconsisting of: Grave's opthalmopathy, epiretinal fibrosis, retinalfibrosis, subretinal fibrosis, subretinal fibrosis associated withmacular degeneration (AMD), subretinal fibrosis associated with wet AMD,diabetic retinopathy, glaucoma, corneal fibrosis, post-surgicalfibrosis, fibrosis of the posterior capsule following cataract surgery,fibrosis of the bleb following trabeculectomy for glaucoma, conjunctivalfibrosis, and subconjunctival fibrosis in a human subject, the methodcomprising administering to the human subject in need of treatment atherapeutically effective amount of an Interleukin 11 receptor α(IL-11Rα) antibody which is capable of inhibiting Interleukin 11 (IL-11)mediated signaling.
 8. The method according to claim 7, wherein theantibody is capable of inhibiting or reducing the binding of IL-11 to anIL-11 receptor.
 9. The method according to claim 7, wherein the methodcomprises administering said antibody to a subject in which IL-11 orIL-11R expression is upregulated.
 10. The method according to claim 7,wherein the method comprises administering said antibody to a subject inwhich IL-11 or IL-11R expression has been determined to be upregulated.11. The method according to claim 7, wherein the method comprisesdetermining whether IL-11 or IL-11R expression is upregulated in thesubject and administering said antibody to a subject in which IL-11 orIL-11R expression is upregulated.